Systems and methods for two-dimensional chromatography

ABSTRACT

Provided are two-dimensional chromatography systems and methods for separating and/or analyzing complex mixtures of organic compounds. In particularly, a two-dimensional reversed-phase liquid chromatography (RPLC)—supercritical fluid chromatography (SFC) system is described including a trapping column at the interface which collects the analytes eluted from the first dimension chromatography while letting the RPLC mobile phase pass through. The peaks of interest from the RPLC dimension column are effectively focused as sharp concentration pulses on the trapping column, which is subsequently injected onto the second dimension SFC column. The system can be used for simultaneous achiral and chiral analysis of pharmaceutical compounds. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/923,273, now U.S. Pat. No. 10,101,306, filed Oct. 26, 2015, whichclaims priority to U.S. Provisional Patent Application No. 62/069,219,filed Oct. 27, 2014, the disclosures of each of which are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to multidimensional chromatography systems andmethods for orthogonal separation and analysis of mixtures of compounds.Disclosed are exemplary two-dimensional reversed-phased liquidchromatography-supercritical fluid chromatography systems and methods ofuse thereof.

BACKGROUND OF THE INVENTION

Chromatography is widely used in separation and analysis of mixtures ofcompounds. Due to limitations in peak capacity of one-dimensionalchromatography, multi-dimensional chromatography systems withsignificantly increased peak capacity have been devised for the analysisof complex samples. Two-dimensional (2D) chromatographic techniques havebecome very popular especially in the analysis of complex mixtures. Ascompared to one-dimensional (1D) chromatography, 2D chromatographictechniques have higher selectivity and resolving power assuming theretention mechanisms are complementary. Maximum peak capacity in atwo-dimensional separation system is achieved when the selectivity ofthe individual separations are independent (orthogonal), so thatcomponents which are poorly resolved in the first dimension may becompletely resolved in the second dimension. If orthogonal separationmechanisms are used in the two dimensions, the theoretical peak capacityof the system is the product of the individual peak capacities. SeeGiddings, J. C. Anal. Chem. 1984, 56:1258A; Giddings, J. C., in: Cortes,H. J. (Ed.), Multidimensional Chromatography: Techniques andApplications, Marcel Dekker, New York 1990, p. 1; Jandera, P. et al.,Chromatographia 2004, 60:S27.

However, some restrictions exist for 2D chromatography in terms ofsensitivity and solvent compatibilities. For example, the mobile phasethat is carried over from the first dimension often creates interferencewith the second dimension, thus limiting the separation capability ofthe second dimension. The incompatibility of solvents used in the firstand second dimensions can cause severe band dispersion or broadening andpeak deterioration, thus posing a big challenge for the interfacedesign. Tian, H., et al., J. Chromatogr. A 2006, 1137:42. To alleviatethe solvent immiscibility concern, researchers have developed several 2Dsystems that use compatible mobile phases in both dimensions. Someexamples include the following: 2D Reversed Phase Liquid Chromatography(RPLC×RPLC) (Venkatramani, C. J. et al., J. Sep. Sci. 2012, 35:1748;Zhang, J. et al., J. Sep. Sci. 2009, 32:2084; Song, C. X. et al., Anal.Chem. 2010, 82:53; Liu, Y. M. et al., J. Chromatogr. A 2008, 1206:153),2D Hydrophilic Interaction Liquid Chromatography (HILIC×RPLC) (Liu, Y.M. et al., J. Chromatogr. A 2008, 1208:133; Wang, Y. et al., Anal. Chem.2008, 80:4680; Louw, S. et al., J. Chromatogr. A 2008, 1208:90), 2DNormal Phase Liquid Chromatography×Supercritical Fluid Chromatography(NPLC×SFC) (Gao, L. et al., J. Sep. Sci. 2010, 33:3817), and 2D SFC×SFC(Zeng, L. et al., J. Chromatogr. A 2011, 1218:3080; Lavison, G. et al.,J. Chromatogr. A 2007, 1161:300; Hirata, Y. et al., J. Sep. Sci. 2003,26:531; Okamoto, D. et al., Anal. Sci. 2006, 22:1437; Guibal, P. et al.,J. Chromatogr. A 2012, 1255:252). Anderer et al., US 2013/0134095disclosed a 2D LC system and methods which attempt to further reduce theinterference with the second LC by the first LC by controlling theinjection event of injecting an output of the first LC into the secondLC in relation to the state of the second LC.

One technique that couples the incompatible “normal phase” and“reversed-phase” dimensions is 2D SFC×RPLC (Francois, I. et al., J. Sep.Sci. 2008, 31:3473-3478; Francois, I. and Sandra, P. J. Chromatogr. A2009, 1216:4005). In this case, the non-polar supercritical carbondioxide in the SFC fractions is evaporated off (when exposed toatmospheric pressure) to yield fractions with compatible mobile phasesto the second RPLC dimension (usually an alcohol modifier). The SFC×RPLCsystem of Francois, I. et al. employs a 2-position/10-port switchingvalve for the interface between the first dimension SFC unit and thesecond dimension RPLC unit. The system uses packed loops in theinterface to prevent the analytes eluted from the SFC column from beingforced into the waste line by the CO₂ stream, and water is introducedinto the loop to reduce interference of residual CO₂ gas in the seconddimension.

Cortes and co-workers (J. Microcol. Sep. 1992, 4:239-244; and U.S. Pat.No. 5,139,681) described a 2D LC×SFC system including a sample inletcapillary where volatile solvents from the first dimension LC iseliminated by passage of nitrogen gas leaving a deposit of the elutedanalytes, which is then taken up by the CO₂ mobile phase for the seconddimension SFC. However, solvent elimination by passage of nitrogen gasis not practical for RPLC which uses aqueous mobile phase.

There remains a need for an efficient chromatography system and methodsfor separating and analyzing complex samples where it is advantageous toconduct a first dimension RPLC and a second dimension SFC separation.

BRIEF SUMMARY OF THE INVENTION

Disclosed are multi-dimensional chromatography systems and methods forseparating and/or analyzing complex mixtures of organic compounds, forexample, a two-dimensional reversed-phase liquid chromatography(RPLC)—supercritical fluid chromatography (SFC) system, particularly aRPLC×SFC system including an interface capable of retaining the analyteseluted from the RPLC column while letting the RPLC mobile phase passthrough, thus reducing the amount of RPLC solvents carried on to the SFCcolumn.

In one aspect, provided is a chromatography system for separating asample comprising: (i) a first separation unit comprising: a) a firstpump assembly for driving a first mobile phase through the firstseparation unit, b) a sample injector for introducing a sample to thefirst separation unit, and c) a reversed-phase liquid chromatography(RPLC) column; (ii) a second separation unit comprising: a) a secondpump assembly for driving a second mobile phase through the secondseparation unit, and b) a supercritical fluid chromatography (SFC)column; and (iii) a first fluidic routing unit comprising a plurality ofsample loops, said first fluidic routing unit is connected to the firstseparation unit and the second separation unit, wherein at least one ofthe plurality of sample loops comprises a trapping column, said trappingcolumn comprising a stationary phase; and wherein the chromatographysystem is configured for first separating the sample in the firstseparation unit and subsequently introducing at least a portion of thesample eluted from the RPLC column to the second separation unit. Insome embodiments, the system further comprises detector(s) for the firstseparation unit and/or the second separation unit. The system mayfurther comprise one or more control devices operably connected to oneor more of the system components.

In some embodiments, the 2D RPLC×SFC system comprises a first fluidicrouting unit which comprises two sample loops; wherein one of the twosample loops is in fluidic communication with the first separation unitand the other one of the two sample loops is in fluidic communicationwith the second separation unit. In some embodiments, the first fluidicrouting unit comprises three or more sample loops, and wherein one ormore of the sample loops is in fluidic isolation from the firstseparation unit and the second separation unit. In one variation, atleast one sample loop that is in fluidic isolation from the firstseparation unit and the second separation unit comprises a trappingcolumn loaded with a stationary phase material. In some embodiments, thefirst fluidic routing unit is configured to allow countercurrent elutionof analytes retained in a trapping column. In some embodiments, thefirst fluidic routing unit is configured to allow co-current elution ofanalytes retained in a trapping column.

In some embodiments, the 2D RPLC×SFC system comprises a secondseparation unit which comprises one SFC column, and optionally a focuscolumn positioned upstream of the SFC column. In some embodiments, thesecond separation unit comprises a parallel array of SFC columns, eachoptionally comprise a focus column positioned upstream of the SFCcolumn.

Further provided are methods of using the chromatography systemsdescribed herein. In some embodiments, provided is a method foranalyzing a samples (such as a complex sample) using a chromatographysystem described herein comprising: first separating the complex sampleinto fractions by RPLC; and further separating the fractions from theRPLC dimension by SFC in the second dimension.

In one embodiment, provided is a method for simultaneous achiral-chiralanalysis of a sample comprising a mixture of stereoisomeric componentsusing a chromatography system described herein comprising: separating(or resolving) one or more diastereomeric component(s) of interest inthe sample by RPLC in the first dimension, which provides achiral purityof the sample; and separating (or resolving) the enantiomeric pair(s) ofinterest by SFC in the second dimension in the same analytical run,which further provides chiral purity of the components in the sample.

In another aspect, provided is a method for separating a sample bymulti-dimensional chromatography (such as 2D RPLC×SFC) comprising thesteps of: (i) capturing at least a portion of a sample on a trappingcolumn, said portion is from separating the sample by reversed-phaseliquid chromatography (RPLC), said trapping column comprising astationary phase; and (ii) subjecting the portion of the sample capturedon the trapping column to further separation by supercritical fluidchromatography (SFC).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an exemplary multidimensional chromatographysystem 10.

FIG. 2A is a schematic of an exemplary first separation unit 20,interfaced with a first fluidic routing unit 30.

FIG. 2B is a schematic of an exemplary second separation unit 40A and40B interfaced with a first fluidic routing unit 30.

FIG. 3A and FIG. 3B are schematics of an exemplary first fluidic routingunit 30 comprising a trapping column 330, wherein the configuration ofthe first fluidic routing unit 30 is configured for countercurrent flowof the first mobile phase and second mobile phase through said trappingcolumn 330.

FIG. 3C and FIG. 3D are schematics of an exemplary first fluidic routingunit 30 comprising a trapping column 370, wherein the configuration ofthe first fluidic routing unit 30 is configured for co-current flow ofthe first mobile phase and second mobile phase through said trappingcolumn 370.

FIG. 4A and FIG. 4B are schematics of an exemplary first fluidic routingunit 30 comprising a trapping column 430, wherein the configuration ofthe first fluidic routing unit 30 is configured for co-current flow ofthe first mobile phase and second mobile phase through said trappingcolumn 430.

FIG. 5A and FIG. 5B are schematics of an exemplary first fluidic routingunit 30 comprising two trapping columns 530 and 560, wherein theconfiguration of the first fluidic routing unit 30 is configured forcountercurrent flow of the first mobile phase and second mobile phasethrough each of the trapping columns 530 and 560.

FIGS. 6A-6D are schematics of an exemplary first fluidic routing unit 30comprising two routing mechanisms 600 and 620.

FIG. 7A and FIG. 7B are schematics of an exemplary downstream subunit ofthe second separation unit 40B comprising an array of SFC columns.

FIG. 8A and FIG. 8B depict the home/analysis position and the trappingposition, respectively, of an exemplary 2D RPLC×SFC system with onetrapping column (co-current flow).

FIG. 9 shows a schematic diagram of 2D RPLC-SFC using an array oftrapping columns, focusing columns, and secondary SFC columns.

FIG. 10 is a photographic image of an exemplary parking deck valve.

FIG. 11 is an overlay chromatogram of the absorbance measurements (mAU)over time (minutes) for the multidimensional separation oftrans-stilbene oxide (TSO) using a system with three sample loopconfigurations (6 μL, 12 μL, 24 μL).

FIG. 12 is an overlay chromatogram of the UV absorbance measurements(mAU) over time (minutes) for the multidimensional separation of anevaluated concentration range (0.005-0.25 mg/ml) of TSO.

FIG. 13 is an overlay chromatogram of unspiked and spiked analyses of asample of Drug Substance A obtained from a 2D RPLC-SFC system.

FIG. 14A and FIG. 14B are conventional SFC and 2D RPLC-SFCchromatograms, respectively, of a sample of Drug Substance A containingvarying levels of an undesired enantiomer (0.1 to 2.0% range).

FIG. 15 is a series of chromatograms illustrating the separation of 8stereoisomers of a sample of Drug Substance A using a 2D RPLC-SFCsystem. The lower chromatogram shows the separation of 4 diastereomericpairs of a sample of Drug Substance A in the first RPLC dimension. Thefour upper chromatograms demonstrate that each diastereomeric pair wasfurther separated in the second SFC dimension.

FIG. 16 is a plot of peak height versus peak area from analyses of TSOover a concentration range using a 2D RPLC-SFC system with and without afocus column located at the upstream head of the SFC column.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides efficient chromatography systems and methods forseparating and analyzing complex samples, particularly two-dimensionalRPLC×SFC systems and methods of use thereof.

The term “a” or “an” as used herein, unless clearly indicated otherwise,refers to one or more.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

Systems

In one aspect, the invention provides a two-dimensional RPLC×SFCchromatography system for separating a sample, wherein the sample isseparated in the first dimension RPLC and then in the second dimensionSFC, the system comprising a first separation unit for reversed-phaseliquid chromatography, a second separation unit for supercritical fluidchromatography, and an interface which is a fluidic routing unit. Thefluidic routing unit comprises multiple sample loops each having apredefined volume that can be placed in the fluidic path of the firstseparation unit for collecting fractions eluted from the RPLC column. Asample loop in which a fraction has been collected is subsequentlyplaced in the fluidic path of the second separation unit fortransferring the fraction collected in the sample loop onto the SFCcolumn for further separation. In a system of the present invention, atleast one of the sample loops comprises a trapping column containing astationary phase for retaining the analytes eluted from RPLC columnwhile letting the mobile phase pass through. The new interface designallows the coupling of RPLC and SFC.

In some embodiments, provided is an online two-dimensionalchromatographic system utilizing RPLC in the first dimension and SFC inthe second dimension, which can achieve simultaneous achiral and chiralanalysis of pharmaceutical compounds. In certain embodiments, theinterface comprises a 2-position/8-port switching valve with smallvolume C-18 trapping columns. The peaks of interest from the first RPLCdimension column were effectively focused as sharp concentration pulseson small volume trapping column(s) (e.g., C-18 trapping column(s)) andthen injected onto the second dimension SFC column. The first dimensionRPLC separation provides the achiral purity result, and the seconddimension SFC separation provides the chiral purity result (enantiomericexcess).

Referring to the drawings, FIG. 1 depicts an overview schematic of anexemplary multidimensional chromatography system 10. A first separationunit 20 and a second separation unit 40A and 40B are interfaced with afirst fluidic routing unit 30. The directional flow of a first mobilephase through the first separation unit 20 to the first fluidic routingunit 30 is indicated by the arrow 50. A second mobile phase, thedirectional flow indicated by the arrow 60, travels through the upstreamsubunit 40A of the second separation unit to the first fluidic routingunit 30 and is subsequently directed back through downstream subunit 40Bof the second separation unit in the direction indicated by the arrow70.

In some embodiments, the system comprises one or more control deviceoperably connected to one or more components of the system forcontrolling the operation of the system, for example, the pumpassemblies, the sample injector, the first fluidic routing unit(interface), and the detectors that are present. The control device mayinclude a computer system equipped with appropriate software forcontrolling the operation of each of the individual devices and forautomated sample analysis (e.g. Agilent's Instrument Control Software orAutomation Studio software). Referring to FIG. 1, a control device 80 isoperably connected to at least one of the following: the firstseparation unit 20, the first routing unit 30, and the second separationunit 40A and 40B.

The composition of the mobile phase in chromatography may be keptconstant over time, what is known in the art as isocratic mode.Alternatively, the composition of the mobile phase may be varied overtime, what is known in the art as gradient mode.

The first separation unit of the 2D RPLC×SFC chromatography systemcomprises a first pump assembly for driving a mobile phase through thefirst separation unit, a sample injector for introducing a sample to thefirst separation unit, and a reversed-phase liquid chromatography (RPLC)column. The mobile phase for RPLC may comprise a two-solvent system(e.g., water-acetonitrile and water-methanol mixtures) and optionallycertain additives (e.g., acetic acid, trifluoroacetic acid, formic acid,ammonium hydroxide, ammonium acetate, sodium acetate, and the like). Thepump assembly comprises one or more pumps for driving a mobile phase forRPLC. While suitable pumps for liquid chromatography are readily knownin the art, in some embodiments, the pump may be a reciprocating pump, adisplacement pump, a pneumatic pump, and/or any combination of the atleast one of the above. The system may comprise any suitable sampleinjector for injecting a sample into the mobile phase for RPLC, such asa manual sample injector or an auto-sampler. The RPLC column comprises areversed-phase stationary phase such as beads of C-18 stationary phase.The reversed-phase stationary phase can be silica-based (for examplecontaining a core structure of silica gel) or polymer-based (for examplecontaining a core structure of an organic polymer (e.g., polystyrene)).Non-limiting examples of materials suitable for reversed-phasestationary phase includes C-18, C-8, C-4, phenyl and phenyl derivatives,polar embedded phase, mixed mode phase, and the like.

In some embodiments, the first separation unit further includes adetector positioned downstream of the RPLC column to detect presence ofthe analytes eluted from the RPLC column. Any detector suitable fordetecting the compounds in the sample may be used, such as adifferential refractometer, ultraviolet spectrophotometer,ultraviolet-visible spectrophotometer detector, charged aerosoldetector, fluorescence detector, and mass spectrometer. In someembodiments, the detector for the RPLC dimension is anultraviolet-visible spectrophotometer detector, charged aerosoldetector, fluorescence detector, and mass spectrometer.

The detector may be optional in a system where the analytes are elutedat a predetermined time, for example, when the RPLC is run underpre-programmed conditions and the retention time for the analytes ofinterests are pre-determined.

Referring to the drawings, FIG. 2A shows a schematic of an exemplaryfirst separation unit 20 interfaced with the first fluidic routing unit30. The first separation unit 20 comprises a first pump 100 for drivingthe first mobile phase through the first separation unit in thedirection indicated by the arrow 110. In some embodiments, the firstmobile phase may be comprised of only one solvent. In some embodiments,the first mobile phase may be comprised of a plurality of mixedsolvents. In some embodiments, mixing of solvents of the first mobilephase may be provided upstream of the pump 100 so that the pump 100receives the mixed solvent as the first mobile phase. In someembodiments the first mobile phase is stored in a receptacle 120. Insome embodiments, the pump 100 may be comprised of a plurality ofindividual pumping units, with plural of the pumping units eachreceiving and pumping a different solvent or mixture of solvents so thatmixing occurs downstream of the pump 100. In some embodiments, the firstmobile phase may further comprise one or more additives. Non-limitingexamples of additives used in RPLC mobile phase include phosphoric acid,phosphate buffers, acetic acid, trifluoroacetic acid, formic acid,ammonium hydroxide, ammonium acetate, sodium acetate, alkyl sulfonates,and the like.

In some embodiments, the composition of the first mobile phase may bekept constant over time (running in isocratic mode). In someembodiments, the composition of the first mobile phase may be variedover time (running in gradient mode). Operation in the gradient modetypically requires two pumps—one for driving the more polar solvent inthe mobile phase (e.g. water); the other for driving the less polarsolvent (e.g., acetonitrile or methanol). Operation in the isocraticmode may employ a system with two pumps delivering two solvents at aconstant ratio, or a system with one pump driving a pre-mixed mobilephase.

Furthermore, the schematic of FIG. 2A illustrates a sample injector 130located between the pump 100 and a reversed-phase liquid chromatography(RPLC) column 150. The sample injector 130 introduces the sample to thefirst separation unit in the direction indicated by the arrow 140. Insome embodiments, the sample injector 130 is provided to add a sample tothe first separation unit. In some embodiments, the sample injector 130may comprise a valve and a sample loop for introducing the sample to thefirst separation unit. In some embodiments, the sample injector 130 maycomprise an autosampler.

As shown in the schematic of FIG. 2A, the RPLC column 150 is locateddownstream of the sample injector 130. The RPLC column 150 may comprisea reversed-phase stationary phase. In some embodiments, the RPLC column150 may comprise a carbon chain-bonded silica gel. In some embodiments,the carbon chain-bonded silica gel may comprise a carbon chain that is18 carbons in length (i.e., C18-reversed-phase silica gel). In someembodiments, the carbon chain-bonded silica gel may comprise a carbonchain that is 8 carbons in length (i.e., C8-reversed-phase silica gel).In some embodiments, the carbon chain-bonded silica gel may comprise acarbon chain that is 4 carbons in length (i.e., C4-reversed-phase silicagel). In some embodiments, the reversed-phase stationary phase maycomprise a hydrocarbon chain bonded to a polymer core such as an organicpolymer (e.g., polystyrene).

In some embodiments, the temperature of the RPLC column 150 may bemaintained at a selected temperature. In some embodiments, thetemperature of the RPLC column 150 may be maintained at a range of about10° C. to about 50° C. In some embodiments, the temperature of the RPLCcolumn 150 may be maintained at about 40° C.

Optionally located between the RPLC column 150 of the first separationunit 20 and the first fluidic routing unit 30 is a first detector 160(FIG. 2A). The first detector measures the presence of at least aportion of the sample eluted from the RPLC column 150. In someembodiments, the first detector 150 is an optical detector. In someembodiments, the first detector 150 is a spectrophotometric detector. Insome embodiments, the first detector 150 is selected from one or more ofthe following: ultraviolet spectrophotometer, ultraviolet-visiblespectrophotometer detector and fluorescence detector.

The second separation unit of the 2D RPLC×SFC chromatography systemcomprises a second pump assembly for driving a second mobile phasethrough the second separation unit and a supercritical fluidchromatography (SFC) column. The mobile phase for SFC comprises asupercritical fluid (e.g., supercritical carbon dioxide) and a modifieror co-solvent (e.g., methanol, ethanol, and isopropyl alcohol),optionally one or more additives (e.g., ammonium hydroxide). The secondpump assembly comprises one or more pumps for driving the supercriticalfluid mobile phase. Any stationary phase suitable for SFC may be used inthe SFC column. The choice of the stationary phase material may dependon the separation criteria for the second dimension. In someembodiments, the SFC column comprises a normal phase stationary phasesuch as silica gel. In some embodiments, the chromatography systemfurther comprises a detector positioned downstream of the SFC column fordetecting the presence of analytes eluted from the SFC column. Anydetectors suitable for SFC may be used, such as a UV detector, aphotodiode array detector, charged aerosol detector, fluorescencedetector, and a mass spectrometer (MS).

Referring to the drawings, FIG. 2B depicts a schematic of an exemplarysecond separation unit, comprising an upstream subunit 40A and adownstream subunit 40B, interfaced with the first fluidic routing unit30. The upstream subunit 40A of the second separation unit comprises asecond pump 200 for driving a second mobile phase through the secondseparation unit 40A and 40B in the direction indicated by the arrow 210.The downstream subunit 40B of the second separation unit comprises anSFC column 240, and optionally a focus column 260 and/or a detector 250.In some embodiments, the second mobile phase may be comprised of onlyone solvent. In some embodiments, the second mobile phase may becomprised of a plurality of mixed solvents. In some embodiments, mixingof solvents of the second mobile phase may be provided upstream of thepump 200 so that the pump 200 receives the mixed solvents as the secondmobile phase. In some embodiments, the second mobile phase is stored ina receptacle 220. In some embodiments, the pump 200 may be comprised ofa plurality of individual pumping units, with plural of the pumpingunits each receiving and pumping a different solvent or mixture ofsolvents so that mixing occurs downstream of the pump 200.

In some embodiments, the composition of the second mobile phase may bekept constant over time (running in isocratic mode). In someembodiments, the composition of the second mobile phase may be variedover time (running in gradient mode). While suitable pumps forsupercritical fluid chromatography are readily known in the art, in someembodiments, the pump may be a reciprocating pump, a displacement pump,a pneumatic pump, and/or any combination of the at least one of theabove.

As shown in FIG. 2B, the upstream subunit 40A of the second separationunit is interfaced with the first fluidic routing unit 30 downstream ofthe pump 200. The directional flow of the second mobile phase throughthe downstream subunit 40B of the second separation unit downstream ofthe first fluidic routing unit 30 is shown by the arrow 230. The SFCcolumn 240 may comprise a normal phase stationary phase. In someembodiments, the normal phase stationary phase is silica. In someembodiments, the normal phase stationary phase is silica modified withcyanopropyl functional groups. In some embodiments, the normal phasestationary phase is silica modified with aminopropyl functional groups.In some embodiments, the normal phase stationary phase is silicamodified with ethyl pyridine functional groups. In some embodiments, thenormal phase stationary phase is silica modified with sulfonic acidand/or phenyl functional groups. In some embodiments, the normal phasestationary phase is silica modified with 1,2-dihydroxypropyl propylether functional groups. In some embodiments, the normal phasestationary phase is a polymer, such as an organic polymer (e.g.,polystyrene), modified with a functional group (e.g., a cyanopropyl,aminopropyl, ethyl pyridine, sulfonic acid, phenyl, or1,2-dihydroxypropyl propyl ether functional group). Other examples ofmaterials suitable for use in the stationary phase for SFC includesilica, ethyl pyridine, cyano, epic diol, pyridyl amide, nitro, and thelike.

In some embodiments, the temperature of the SFC column 240 may bemaintained at a selected temperature. In some embodiments, thetemperature of the SFC column 240 may be maintained at a range of about35° C. to about 45° C. In some embodiments, the temperature of the SFCcolumn 240 may be maintained at about 40° C.

Referring to FIG. 2B, in some embodiments, an optional focus column 260is located upstream of the SFC column 240. The focus column 260comprises a stationary phase. In some embodiments, the stationary phasemay comprise a reversed-phase stationary phase. In some embodiments, thereversed-phase stationary phase may comprise a carbon chain-bondedsilica gel. In some embodiments, the carbon chain-bonded silica gel maycomprise a carbon chain that is 18 carbons in length (i.e.,C18-reversed-phase silica gel). In some embodiments, the carbonchain-bonded silica gel may comprise a carbon chain that is 8 carbons inlength (i.e., C8-reversed-phase silica gel). In some embodiments, thecarbon chain-bonded silica gel may comprise a carbon chain that is 4carbons in length (i.e., C4-reversed-phase silica gel). In someembodiments, the reversed-phase stationary phase may comprise a carbonchain (such as a C-18, C-8 or C-4 chain) bonded to a polymer core suchas an organic polymer (e.g., polystyrene). In general focus column willmatch the reverse phase used in the primary dimension and/or thetrapping column used in the interface.

In some embodiments, the temperature of the focus column 260 may bemaintained at a selected temperature. In some embodiments, the focuscolumn may be maintained at the same temperature as the SFC column. Insome embodiments, the temperature of the focus column 260 may bemaintained at about 35° C. to about 45° C. In some embodiments, thetemperature of the focus column 260 may be maintained at about 40° C.

Located downstream of the SFC column 240 is a second detector 250 (FIG.2B). The detector measures the presence and amounts of analytes elutedfrom the SFC column 240. In some embodiments, the second detector 250 isan optical detector. In some embodiments, the second detector 250 is aspectrophotometric detector. In some embodiments, the second detector250 is selected from one or more of the following: differentialrefractometer, ultraviolet spectrophotometer, ultraviolet-visiblespectrophotometer detector, fluorescence detector, and infraredspectrophotometer. In some embodiments, the second detector 250 is amass spectrometer. In some embodiments, the mass spectrometer may beselected from one or more of the following: a sector instrument, aquadrupole mass filter instrument, a time-of-flight instrument, anion-trap instrument, a quadrupole ion trap instrument, a linearquadrupole ion trap instrument, an orbitrap instrument, a Fouriertransform ion cyclotron resonance instrument, and any combination orhybrid of the listed instrument types. As is well known in the art, thesample is introduced into the mass spectrometer via ionizationtechniques, such as, but not limited to, fast atom bombardment, chemicalionization, atmospheric-pressure chemical ionization, electrosprayionization, and nano-electrospray ionization. In some embodiments, thesample is introduced into the mass spectrometer via atmospheric-pressurechemical ionization or electrospray ionization.

In some embodiments, the downstream portion of the second separationunit 40B may further comprise a fraction collector in place, after, orin parallel with the second detector.

Optionally, in some embodiments, a control device 80 is operablyconnected to at least one of the following: a pump 100 and/or 200, asample injector 130, a first detector 160, the first fluidic routingunit 30, and a second detector 250.

The interface between the two dimensions in a 2D chromatography systemcontrols routing of analytes separated and eluted from the firstdimension into the second dimension for further separation anddetermines the mode of operation. The 2D chromatography system of thepresent invention is configured for first separating the sample in thefirst separation unit by RPLC and subsequently introducing at least aportion of the sample eluted from the RPLC column to the secondseparation unit for further separation by SFC.

The first fluidic routing unit comprises a plurality of sample loops,said first fluidic routing unit is connected to the first separationunit and the second separation unit, wherein at least one of theplurality of sample loops comprises a trapping column, said trappingcolumn comprising a stationary phase. The stationary phase material inthe trapping columns may be the same as the stationary phase materialused in the RPLC column or different from the stationary phase materialused in the RPLC column. In some embodiments, the stationary phase usedin the trapping column may comprise a reversed-phase stationary phase.In some embodiments, the reversed-phase stationary phase may comprise acarbon chain-bonded silica gel. In some embodiments, the carbonchain-bonded silica gel may comprise a carbon chain that is 18 carbonsin length (i.e., C18-reversed phase silica gel). In some embodiments,the carbon chain-bonded silica gel may comprise a carbon chain that is 8carbons in length (i.e., C8-reversed phase silica gel). In someembodiments, the carbon chain-bonded silica gel may comprise a carbonchain that is 4 carbons in length (i.e., C4-reversed phase silica gel).In some embodiments, the reversed-phase stationary phase may comprise acarbon chain (such as a C-18, C-8 or C-4 chain) bonded to a polymer coresuch as an organic polymer (e.g., polystyrene).

The first fluidic routing unit in some embodiments includes a routingmechanism (such as a switch valve) comprising a plurality of ports,channels allowing liquid to flow between the ports and sample loopsconnected to the ports. In some embodiments, the routing mechanism is a2-position/8-port switching valve. In some embodiments, the routingmechanism is a 2-position/10-port switching valve. In some embodiments,the routing mechanism is a 2-position/4-port duo valve. In someembodiments, the routing mechanism is a 2-position 8-port or 10-portswitching valve.

In some embodiments, the first fluidic routing unit of the 2D RPLC×SFCsystem comprises two sample loops; wherein one of the two sample loopsis in fluidic communication with the first separation unit and the otherone of the two sample loops is in fluidic communication with the secondseparation unit. In some embodiments, one of the two sample loopscomprises a trapping column comprising a stationary phase. In someembodiments, both sample loops each comprise a trapping columncomprising a stationary phase.

FIG. 3A depicts a schematic of an exemplary first fluidic routing unit30 interfaced with the first separation unit 20 and the secondseparation unit 40A and 40B. In this instance, the interface fluidicrouting unit includes a routing mechanism 300, which in one variation isa 2-positio/8-port switching valve, comprising a plurality of ports305A-305H, a plurality of channels 310A-310D, and two sample loops, 315and 320. As shown in FIG. 3A, the plurality of channels 310A-310D areshown in the first of two possible configurations.

As exemplified in FIG. 3A, the first separation unit 20 is connected tothe routing mechanism 300 at port 305A. The first mobile phase is driventhrough the first separation unit 20 in the direction indicated by thearrow 325 and the routing mechanism 300 directs the first mobile phaseto a sample loop 315 by way of the channel 310A. The sample loop 315 isconnected to the routing mechanism 300 at port 305B and port 305F. Thesample loop 315 comprises a trapping column 330, said trapping columncomprises a stationary phase.

The routing mechanism 300 directs the first mobile phase to a downstreamreceptacle 340, in the direction indicated by the arrow 345, by way of achannel 310C. In some embodiments, the downstream receptacle 340 is awaste receptacle. In some embodiments, the downstream receptacle 340 isa fraction collector.

As depicted in FIG. 3A, the upstream subunit 40A of the secondseparation unit is connected to the routing mechanism 300 at port 305G.The second mobile phase is driven through the second separation unit 40Ain the direction indicated by the arrow 350 and the routing mechanism300 directs the second mobile phase to a sample loop 320 by way of achannel 310D. The sample loop 320 is connected to the routing mechanism300 at port 305H and port 305D, wherein the first mobile phase travelsin the direction indicated by the arrow 355. The routing mechanism 300is connected to the downstream subunit 40B of the second separation unitvia port 305C. The routing mechanism 300 directs the second mobile phasethrough the downstream subunit 40B of the second separation unit in thedirection indicated by the arrow 360.

The schematic illustrated in FIG. 3B depicts the same configuration ofthe first fluidic routing unit as shown in FIG. 3A, except the pluralityof channels 310A-310D of the routing mechanism 300 in FIG. 3A are now ina second of two of possible positions, namely, channels 310E-310H, asshown in FIG. 3B. The routing mechanism in FIG. 3B directs the firstmobile phase from the first separation unit 20, in the directionindicated by the arrow 325, through the channel 310H to the sample loop320. The sample loop 320 is connected to the routing mechanism 300 atport 305H and port 305D wherein the first mobile phase is directed tothe downstream receptacle 340, in the direction indicated by the arrow345, via channel 310F.

As depicted in FIG. 3B, the upstream subunit 40A of the secondseparation unit is connected to the routing mechanism 300 at port 305G.The second mobile phase is driven through the second separation unit 40Ain the direction indicated by the arrow 350 and the routing mechanism300 now directs the second mobile phase to the sample loop 315 by way ofa channel 310G. The sample loop 315 is connected to the routingmechanism at port 305B and port 305F. The routing mechanism 300 directsthe flow of the second mobile phase through the trapping column 330 inthe direction indicated by the arrow 365. As compared to FIG. 3A, therouting mechanism 300 as shown in FIG. 3B directs the flow of the secondmobile phase through the sample loop 315 in a direction that is oppositeof the flow of the first mobile phase through the same sample loop 315shown in FIG. 3A. This exemplified configuration of the routingmechanism 300 directs the flow of the mobile phases through the sampleloop 315 in what is known in the art as a “countercurrent” manner.

The exemplary schematics illustrated in FIG. 3C and FIG. 3D depict asimilar configuration of the first fluidic routing unit shown in FIG. 3Aand FIG. 3B, except, as shown in FIG. 3C and FIG. 3D, the trappingcolumn 370 is located on sample loop 320. The routing mechanism 300directs the flow of the first mobile phase through the sample loop 320comprising the trapping column 370 in the direction indicated by thearrow 355 (FIG. 3C). In FIG. 3D, the plurality of channels 310A-310D ofthe routing mechanism 300 are in the second of two possible positions.The routing mechanism 300 directs the second mobile phase from upstreamsubunit 40A of the second separation unit through the sample loop 320comprising the trapping column 370 and through the downstream portion ofthe second separation unit 40B in the direction indicated by the arrows355 and 360. As exemplified in FIG. 3C and FIG. 3D flow of the firstmobile phase and second mobile phase travel through the trapping column370 in the same direction, known in the art as a “co-current” manner.

In the instance of a system using a 2-position/8-port switching valve(300) in the interface fluidic routing unit, theco-current/countercurrent configurations can be controlled by theplacement of the trapping column (370) in sample loop 315 or 320.Alternatively, the co-current/countercurrent configurations can bechanged by switching the port of connection for the upstream subunit 40Aand the downstream subunit 40B of the second separation unit to theinterface fluidic routing unit.

FIG. 4A depicts a schematic of an exemplary first fluidic routing unit30 interfaced with the first separation unit 20 and the secondseparation unit 40A and 40B. In this instance, the interface fluidicrouting unit includes a routing mechanism 400, which in one variation isa 2-position/10-port switching valve, comprising a plurality of ports405A-405J, a plurality of channels 410A-410E, and two sample loops 415and 420. As shown in FIG. 4A, the plurality of channels 410A-410E areshown in the first of two possible configurations.

As exemplified in FIG. 4A, the first separation unit 20 is connected tothe routing mechanism 400 at port 405A. The routing mechanism 400directs the first mobile phase to one of the plurality of sample loops415 by way of the channel 410A in the direction indicated by the arrow425. The sample loop 415 is connected to the routing mechanism 400 atport 405B and port 405E. The sample loop 415 comprises a trapping column430, said trapping column comprises a stationary phase.

The routing mechanism 400 directs the first mobile phase to a downstreamreceptacle 440 by way of a channel 410C. In some embodiments, thedownstream receptacle 440 is a waste receptacle. In some embodiments,the downstream receptacle 440 is a fraction collector.

As depicted in FIG. 4A, the second separation unit 40 is connected tothe routing mechanism 400 at port 405C. The routing mechanism 400directs the second mobile phase to a sample loop 420 by way of twoconnected channels 410D and 410E. The sample loop 420 is connected tothe routing mechanism 400 at port 405J and port 405G. The routingmechanism 400 is connected to the downstream subunit 40B of the secondseparation unit via port 405H. The routing mechanism 400 directs thesecond mobile phase through the downstream portion of the second fluidicrouting unit 40B in the direction indicated by the arrow 435.

The schematic illustrated in FIG. 4B depicts the same configuration ofthe first fluidic routing unit as shown in FIG. 4A, except the pluralityof channels 410A-410E of the routing mechanism 400 in FIG. 4A are now ina second of two of possible positions, namely, channels 410F-410J, asshown in FIG. 4B. As depicted in FIG. 4A and FIG. 4B, the routingmechanism 400 of the first separation unit 30 may be configured so thatdirection of the flow of the first mobile phase through a sample loop415 (FIG. 4A) is in the same direction, indicated by the arrow 425, asthe flow of the second mobile phase through the same sample loop 415(FIG. 4B). This exemplified configuration of the routing mechanism 400directs the flow of the mobile phases through the sample loop 415 in theco-current manner.

In the instance of a system using a 2-position/10-port switching valve(400) in the interface fluidic routing unit, both sample loops areconfigured in the co-current manner as depicted in FIG. 4A and FIG. 4B.However, both sample loops can be configured to countercurrent manner byswitching the port of connection for the upstream subunit 40A and thedownstream subunit 40B of the second separation unit to the interfacefluidic routing unit.

FIG. 5A depicts a schematic of an exemplary first fluidic routing unit30 interfaced with the first separation unit 20 and the secondseparation unit 40A and 40B. In this instance, the interface fluidicrouting unit includes a routing mechanism 500, which in one variation isa 2-position/4-port duo valve, comprising a plurality of ports505A-505H, a plurality of channels 510A-510D, and two sample loops, 515and 520. As shown in FIG. 5A, the plurality of channels 510A-510D areshown in the first of two possible configurations.

As exemplified in FIG. 5A, the first separation unit 20 is connected tothe routing mechanism 500 at port 505G. The first mobile phase is driventhrough the first separation unit 20 in the direction indicated by thearrow 525 and the routing mechanism 500 directs the first mobile phasethrough a sample loop 520 by way of a channel 510A. The sample loop 520is connected to the routing mechanism 500 at port 505H and port 505B.The routing mechanism 500 directs the first mobile phase through thesample loop 520 in the direction indicated by the arrow 535. The sampleloop 520 comprises a trapping column 530, said trapping column comprisesa stationary phase.

The routing mechanism 500 directs the first mobile phase to a downstreamreceptacle 540, in the direction indicated by the arrow 545, by way of achannel 510B. In some embodiments, the downstream receptacle 540 is awaste receptacle. In some embodiments, the downstream receptacle 540 isa fraction collector.

As depicted in FIG. 5A, the upstream subunit 40A of the secondseparation unit is connected to the routing mechanism 500 at port 505E.The second mobile phase is driven through the second separation unit 40Ain the direction indicated by the arrow 550 and the routing mechanism500 directs the second mobile phase to a sample loop 515 by way of achannel 510C. The sample loop 515 is connected to the routing mechanism500 at port 505F and port 505C, wherein the first mobile phase travelsin the direction indicated by the arrow 555. The sample loop 515comprises a trapping column 560, said trapping column comprises astationary phase. The routing mechanism 500 is connected to thedownstream subunit 40B of the second separation unit via port 505D. Therouting mechanism 500 directs the second mobile phase through thedownstream subunit 40B of the second separation unit in the directionindicated by the arrow 565.

In some embodiments, the trapping columns 530 and 560 comprise the samestationary phase. In some embodiments, the stationary phase may comprisea reversed-phase stationary phase. In some embodiments, the trappingcolumns 530 and 560 comprise the different stationary phase.

The schematic illustrated in FIG. 5B depicts the same configuration ofthe first fluidic routing unit as shown in FIG. 5A, except the pluralityof channels 510A-510D of the routing mechanism 500 in FIG. 5A are now ina second of two of possible positions, namely, channels 510E-510G, asshown in FIG. 5B. The routing mechanism 500 in FIG. 5B now directs thefirst mobile phase from the first separation unit 20, in the directionindicated by the arrow 525, through the channel 510E to the sample loop515. The sample loop 515 is connected to the routing mechanism 500 atport 505C and port 505F wherein the first mobile phase is directed tothe downstream receptacle 540, in the direction indicated by the arrow545, via channel 510F. In FIG. 5B, the routing mechanism 500 directs theflow of the first mobile phase through the sample loop 515 in anopposite direction than that of the second mobile phase through the samesample loop 515 in FIG. 5A.

As depicted in FIG. 5B, the upstream subunit 40A of the secondseparation unit is connected to the routing mechanism 500 at port 505E.The second mobile phase is driven through the second separation unit 40Ain the direction indicated by the arrow 550 and the routing mechanism500 now directs the second mobile phase to the sample loop 520 by way ofa channel 510G. The sample loop 520 is connected to the routingmechanism at port 505B and port 505H. The routing mechanism 500 directsthe flow of the second mobile phase through the trapping column 530 inthe direction indicated by the arrow 575. In FIG. 5B, the routingmechanism 500 directs the flow of the second mobile phase through thesample loop 520 in an opposite direction than that of the first mobilephase through the same sample loop 520 in FIG. 5A.

In the instance of a system using a 2-position/4-port duo valve (500) inthe interface fluidic routing unit, both sample loops are configured inthe countercurrent manner as depicted in FIG. 5A and FIG. 5B. However,if desirable, both sample loops can be configured in the co-currentmanner by switching the port of connection for the upstream subunit 40Aand the downstream subunit 40B of the second separation unit to theinterface fluidic routing unit.

In some embodiments, the first fluidic routing unit of the 2D RPLC×SFCsystem comprises at least three sample loops, and wherein at least oneof the sample loops is in fluidic isolation from the first separationunit and the second separation unit. In such a system, one sample loopis in fluidic communication with the first separation unit, one sampleloop is in fluidic communication with the second separation unit, andone or more sample loops are in fluidic isolation from the firstseparation unit and the second separation unit. In some embodiments, atleast one of the sample loops comprises a trapping column, said trappingcolumn comprises a stationary phase. In some embodiments, at least onesample loop which comprises a trapping column, said trapping columncomprises a stationary phase, is in fluidic isolation from the firstseparation unit and the second separation unit. In some embodiments, thefirst fluidic routing unit comprises a plurality of trapping columnseach positioned in a sample loop. In some embodiments, each of thetrapping columns is loaded with the same stationary phase material. Inother embodiments, the stationary phase material may be adapted for theparticular analytes being retained, thus different stationary phasematerials may be used for different fractions eluted from the firstseparation unit. In some embodiments, the stationary phase may comprisea reversed-phase stationary phase. In some embodiments, thereversed-phase stationary phase may comprise a carbon chain-bondedsilica gel. In some embodiments, the carbon chain-bonded silica gel maycomprise a carbon chain that is 18 carbons in length (i.e.,C18-reversed-phase silica gel or C18 silica gel). In some embodiments,the carbon chain-bonded silica gel may comprise a carbon chain that is 8carbons in length (i.e., C8-reversed-phase silica gel or C8 silica gel).In some embodiments, the carbon chain-bonded silica gel may comprise acarbon chain that is 4 carbons in length (i.e., C4-reversed-phase silicagel or C4 silica gel). In some embodiments, the reversed-phasestationary phase may comprise a carbon chain (such as a C-18, C-8 or C-4carbon chain) bonded to a polymer core such as an organic polymer (e.g.,polystyrene).

A system comprising an interface fluidic routing unit where one or moresample loops can be placed in fluidic isolation from the firstseparation unit and the second separation unit provides for a “peakparking” feature. When multiple fractions separated and eluted from thefirst dimension chromatography need to be further separated in thesecond dimension, the time interval between fractions eluted from thefirst separation unit may be insufficient for the first fraction to runthrough the second separation unit. While the second separation unit isin use for analyzing an earlier fraction, later fractions (or cuts) canbe collected on a trapping column in additional loops and retained influidic isolation (“parked”) until the second separation unit is readyfor the analyzing the next fraction.

Referring to the drawings, FIG. 6A depicts a schematic of an exemplaryfirst fluidic routing unit 30 interfaced with the first separation unit20 and the second separation unit 40A and 40B. In this instance, theinterface fluidic routing unit includes a first routing mechanism 600,which in one variation is a 2-position/4-port duo valve, comprising aplurality of ports 605A-605H, a plurality of channels 610A-610D, asample loop 615, and second routing mechanism 620 comprising a pluralityof ports, for example 625A-625E, a plurality of sample loops, forexample, 630A-630C, a plurality of trapping columns, 635A-635E, andplurality of channels 675A and 675B. Sample loop 630B provides for aby-pass loop without a trapping column. As shown in FIG. 6A, theplurality of channels 610A-610D of the first routing mechanism 600 areshown in the first of two possible configurations.

As exemplified in FIG. 6A, the first separation unit 20 is connected tothe routing mechanism 600 at port 605G. The first mobile phase is driventhrough the first separation unit 20 in the direction indicated by thearrow 640 and the routing mechanism 600 directs the first mobile phasethrough a sample loop 615 by way of a channel 610A. The sample loop 615is connected to the first routing mechanism 600 at port 605H and port605B. The first routing mechanism 600 directs the first mobile phasethrough the sample loop 615 in the direction indicated by the arrow 645.As shown in FIG. 6A, the routing mechanism 600 directs the first mobilephase to a downstream receptacle 650, in the direction indicated by thearrow 655, by way of a channel 610B. In some embodiments, the downstreamreceptacle 650 is a waste receptacle. In some embodiments, thedownstream receptacle 650 is a fraction collector.

As depicted in FIG. 6A, the upstream subunit 40A of the secondseparation unit is connected to the routing mechanism 600 at port 605E.The second mobile phase is driven through the upstream subunit 40A ofthe second separation unit in the direction indicated by the arrow 660.The routing mechanism 600 directs the second mobile phase, in thedirection indicated by the arrow 665, to a second routing mechanism 620by way of channel 610C. The first routing mechanism 600 and secondrouting mechanism 620 are connected via port 605F and port 625A.

In FIG. 6A, the second routing mechanism 620 is configured to direct thesecond mobile phase through a sample loop 630B in the directionindicated by the arrow 670 via channel 675A. Sample loop 630B isconnected to the second routing mechanism 620 via port 625B and port625E, which is connected to port 625D via channel 675B. The secondrouting mechanism 620 and first routing mechanism 600 are connected viaport 625F and port 605C. The second routing mechanism 620 directs theflow the second mobile phase back to the first routing mechanism 600 inthe direction indicated by the arrow 680. The first routing mechanism600 directs the flow of the second mobile phase through the downstreamsubunit 40B of the second separation unit via channel 610D in thedirection indicated by the arrow 690.

FIG. 6A depicts a configuration where no trapping column is in fluidiccommunication with either the first separation unit or the secondseparation unit. For example, the system can be set to thisconfiguration when no peak of interest is coming out of the firstdimension column or being analyzed in the second dimension column. Thesystem may also be set to this configuration when the columns in bothseparation units are in idle state or the columns are undergoingregeneration/equilibration. Each one of trapping columns 635A-635E is influidic isolation from the first and second separation unit.

The schematic illustrated in FIG. 6B depicts the same configuration ofthe first fluidic routing unit as shown in FIG. 6A, except the pluralityof channels 610A-610D of the first routing mechanism 600 in FIG. 6A arenow in the second of two possible positions 610E-610H as shown in FIG.6B. The routing mechanism 600 in FIG. 6B now directs the first mobilephase from the first separation unit 20, in the direction indicated bythe arrow 640, through the channel 610E to the second routing mechanism620. The first routing mechanism 600 and the second routing mechanism620 are connected at port 605C and port 625F. Additionally, in FIG. 6B,the second routing mechanism 620 is now configured to direct the flow ofthe first mobile phase through the sample loop 630A in the directionindicated by the arrow 705. Sample loop 630A comprises a trapping column635A. Sample loop 630A is connected to the second routing mechanism 620via port 625F and port 625C. As depicted in FIG. 6B, the second routingmechanism 620 and first routing mechanism 600 are connected via port625A and port 605F. The second routing mechanism 620 directs the flowthe first mobile phase back to the first routing mechanism 600 in thedirection indicated by the arrow 710. The first routing mechanism 600directs the first mobile phase to a downstream receptacle 650, in thedirection indicated by the arrow 655, by way of a channel 610F. In someembodiments, the downstream receptacle 650 is a waste receptacle. Insome embodiments, the downstream receptacle 650 is a fraction collector.In some embodiments, a Flexcube (Agilent) may be used as part of thesecond routing mechanism.

In some embodiments, the configuration of the first fluidic routing unit30 shown in FIG. 6B, may be used to direct a portion of the sampleeluting from the first separation unit to be selectively retained on atrapping column, for example, 635A. In some embodiments, the trappingcolumn may comprise a stationary phase. In some embodiments, thestationary phase may comprise a reversed-phase stationary phase. In someembodiments, the reversed-phase stationary phase may comprise a carbonchain-bonded silica gel (e.g., C18 silica gel, C8 silica gel and C4silica gel). In some embodiments, the reversed-phase stationary phasemay comprise a carbon chain (such as a C-18, C-8 or C-4 carbon chain)bonded to a polymer core such as an organic polymer (e.g., polystyrene).

After a peak has been retained on a trapping column, for example, 635A,the routing mechanisms 600 and 620 can be switched or set to positionsto allow elution and further analysis of the retained peak, for exampleas shown in FIG. 6C; or the routing mechanisms 600 and 620 can beswitched or set to positions to allow parking of the peak retained ontrapping column 635A and collection of another peak on a differenttrapping column, for example, 635B, as demonstrated in FIG. 6D.

The schematic illustrated in FIG. 6C depicts an exemplary configurationof the first fluidic routing unit 30 wherein the first routing mechanism600 is configured to direct the flow of the first mobile phase to thedownstream receptacle 650, as shown in FIG. 6A. As illustrated in FIG.6C, the first mobile phase is driven through the first separation unit20 in the direction indicated by the arrow 640 and the routing mechanism600 directs the first mobile phase through a sample loop 615 by way of achannel 610A. The sample loop 615 is connected to the first routingmechanism 600 at port 605H and port 605B. The first routing mechanism600 directs the first mobile phase through the sample loop 615 in thedirection indicated by the arrow 645. As shown in FIG. 6A, the routingmechanism 600 directs the first mobile phase to a downstream receptacle650, in the direction indicated by the arrow 655, by way of a channel610B. In some embodiments, the downstream receptacle 650 is a wastereceptacle. In some embodiments, the downstream receptacle 650 is afraction collector.

In FIG. 6C, the first routing mechanism 600 directs the flow of thesecond mobile phase to the second fluidic routing mechanism 620 in thedirection indicated by the arrow 665. The second routing mechanism 620is configured to direct the second mobile phase to sample loop 635A viachannel 675D in the direction indicated by the arrow 715. Sample loop635A is connected to the second fluidic routing mechanism via port 625Cand port 625F. The second fluidic routing mechanism 620 is configured todirect the flow of the second mobile phase to the first fluidic routingmechanism 600 via channel 675C in the direction indicated by the arrow680. The second fluidic routing mechanism and the first fluidic routingmechanism are connected via port 625D and 605C and the second mobilephase is directed to the downstream portion 40B of the second separationunit via channel 610D in the direction indicated by the arrow 690.

The schematic illustrated in FIG. 6D depicts the same configuration ofthe first fluidic routing mechanism 600 and the second fluidic routingmechanism 620 as shown in FIG. 6B, except in FIG. 6D, the second fluidicrouting mechanism now directs the flow of the first mobile phase tosample loop 630C in the direction indicated by the arrow 720. Sampleloop 630C is connected to the second fluidic routing mechanism via port625G and port 625H. Channel 675F connects port 625A and port 625G.Channel 675E connects port 625D and port 625H. As depicted in FIG. 6B,sample loop 630C comprises a trapping column 635B, which may comprise astationary phase, such as a stationary phase as described herein.

As exemplified in FIG. 6D, the portion of the sample eluted from thefirst separation unit and selectively retained on the trapping column635A is now in isolation from both the first separation unit 20 andsecond separation unit 40A and 40B.

In some embodiments, the plurality of trapping columns 635A-635E, suchas shown in FIG. 6A-FIG. 6D, may comprise the same stationary phase. Insome embodiments, the stationary phase may comprise a reversed-phasestationary phase. In some embodiments, at least one of the plurality oftrapping columns 635A-635E, such as shown in FIG. 6A-FIG. 6D, maycomprise the same stationary phase as the stationary phase material usedin the first dimension RPLC column. In some embodiments, the stationaryphase may comprise a reversed-phase stationary phase. In someembodiments, the reversed-phase stationary phase may comprise a carbonchain-bonded silica gel. In some embodiments, the carbon chain-bondedsilica gel may comprise a carbon chain that is 18 carbons in length(i.e., C18-reversed-phase silica gel). In some embodiments, the carbonchain-bonded silica gel may comprise a carbon chain that is 8 carbons inlength (i.e., C8-reversed-phase silica gel). In some embodiments, thecarbon chain-bonded silica gel may comprise a carbon chain that is 4carbons in length (i.e., C4-reversed-phase silica gel). In someembodiments, the reversed-phase stationary phase may comprise ahydrocarbon chain (e.g., a C-18, C-8 or C-4 chain) bonded to a polymercore such as an organic polymer (e.g., polystyrene).

2D chromatography can be performed in heart-cutting,pseudo-comprehensive or comprehensive modes. Heart-cutting provides thecharacterization of a selected region of the chromatogram, while thecomprehensive mode provides the characterization of the entirechromatogram. The pseudo-comprehensive mode provides comprehensiveseparation of selected regions of the chromatogram. See Venkatramani, C.J. et al., J. Sep. Sci. 2014, 22, in-press. The interface in the 2DRPLC×SFC system, i.e., the first fluidic routing unit, can be adapted toperform in any one or more of the heart-cutting, pseudo-comprehensiveand comprehensive modes. For example, a 2D RPLC×SFC system having any ofthe interface fluidic routing unit as depicted in FIGS. 3-6 herein maybe used in the heart-cutting mode. A system having an interface fluidicrouting unit comprising two sample loops (each having a trapping column)as depicted in FIGS. 5A and 5B may be used in pseudo-comprehensive modeif a long RPLC column coupled with a slow flow rate of the mobile phasein the first dimension would allow a complete run of one fraction in thesecond dimension separation while another fraction is collected in thesecond sample loop. A system having an interface fluidic routing unitwith the sample parking feature as depicted in FIGS. 6A-6D can be usedin comprehensive mode or pseudo-comprehensive mode as multiple fractionsfrom the first dimension separation can be collected and retained forsubsequent analysis in the second dimension when the second separationunit is available.

The separation criteria in the second dimension may depend on the natureof the analytes to be separated. Different stationary phase materialsmay be required for the SFC column in order to provide optimalseparation for the various analytes eluted from the first dimension RPLCcolumn. Thus, provided is a 2D RPLC×SFC chromatography system describedherein, wherein the second separation unit comprises an array of SFCcolumns, wherein the SFC columns in the array are arranged in a parallelconfiguration; and a second fluidic routing unit for directing flow ofthe second mobile phase to a desired (or pre-identified) SFC column inthe array. In some embodiments, the second separation unit furthercomprises a focus column located upstream of each SFC column in thearray of SFC columns. In some embodiments, the focus column comprises astationary phase. In some embodiments, the stationary phase may comprisea reversed-phase stationary phase. In some embodiments, thereversed-phase stationary phase may comprise a carbon chain-bondedsilica gel. In some embodiments, the carbon chain-bonded silica gel maycomprise a carbon chain that is 18 carbons in length (i.e.,C18-reversed-phase silica gel). In some embodiments, the carbonchain-bonded silica gel may comprise a carbon chain that is 8 carbons inlength (i.e., C8-reversed-phase silica gel). In some embodiments, thecarbon chain-bonded silica gel may comprise a carbon chain that is 4carbons in length (i.e., C4-reversed-phase silica gel). In someembodiments, the reversed-phase stationary phase may comprise ahydrocarbon chain (e.g., a C-18, C-8 or C-4 chain) bonded to a polymercore such as an organic polymer (e.g., polystyrene).

Referring to the drawings, FIG. 7A and FIG. 7B are schematics of anexemplary downstream subunit 40B of the second separation unitcomprising a second fluidic routing unit 800 and an array of SFCcolumns. The first fluidic routing unit 30 is connected to the secondfluidic routing unit 800 at port 805A. The first fluidic routing unit 30directs the second mobile phase to the second routing unit 800 in thedirection indicated by the arrow 810. The second routing unit 800comprises a plurality of ports, such as port 805A and port 805B, and aplurality of channels, such as channel 810A. As depicted in FIG. 7A, thesecond fluidic routing unit 800, directs the second mobile phase to adesired (or pre-identified) SFC column 815A in the direction indicatedby the arrow 825. Optionally, a focus column 820A is located upstream ofthe SFC column 815A.

In some embodiments, a routing mechanism 830 is optionally locateddownstream of the SFC column. As depicted in FIG. 7A, the routingmechanism 830 directs the second mobile phase to a detector 250 viachannel 810B in the direction indicated by the arrow 835.

FIG. 7B depicts the same SFC column array as illustrated in FIG. 7A, butin FIG. 7B the second fluidic routing unit 800 is configured to directthe second mobile phase from the first fluidic routing unit to a seconddesired SFC chromatography column 815B. As depicted in FIG. 7B, thesecond desired SFC chromatography column is connected to the secondfluidic routing unit 800 and the routing mechanism 830 via port 805E andport 805F. The second fluidic routing unit 800 directs the second mobilephase in the direction indicated by the arrow 840. Optionally, a focuscolumn 820B is located upstream of the SFC column 815B.

In some embodiments, the SFC column array comprises a plurality of SFCcolumns wherein an individual SFC column, such as 815A, may comprise thesame stationary phase as at least one other individual SFC column in thearray, such as 815B. In some embodiments, the SFC column array comprisesa plurality of SFC columns wherein an individual SFC column, such as815A, may comprise a different stationary phase from all otherindividual SFC column in the array, such as 815B. In some embodiments,the stationary phase may comprise a normal phase stationary phase. Insome embodiments, the normal phase stationary phase is silica. In someembodiments, the normal phase stationary phase is silica modified withpropylcyano functional groups. In some embodiments, the normal phasestationary phase is silica modified with aminopropyl functional groups.In some embodiments, the normal phase stationary phase is silicamodified with ethyl pyridine functional groups. In some embodiments, thenormal phase stationary phase is silica modified with sulfonic acidand/or phenyl functional groups. In some embodiments, the normal phasestationary phase is silica modified with 1,2-dihydroxypropyl propylether functional groups. In some embodiments, the normal phasestationary phase is a polymer, such as an organic polymer (e.g.,polystyrene), modified with a functional group (e.g., a cyanopropyl,aminopropyl, ethyl pyridine, sulfonic acid, phenyl, or1,2-dihydroxypropyl propyl ether functional group).

In some embodiments, the SFC column array comprises a plurality of focuscolumns wherein an individual focus column, such as 820A, may comprisethe same stationary phase as at least one other individual focus columnin the array, such as 820B. In some embodiments, the SFC column arraycomprises a plurality of focus columns wherein an individual focuscolumn, such as 820A, may comprise a different stationary phase from allother individual focus column in the array, such as 820B.

FIG. 8A and FIG. 8B depict schematics of an exemplary 2D LC-SFC systemwith an interface involving an electronically controlled,2-position/4-port duo valve V1 and trapping column Trap 1. The mobilephase from the pump 1 flows through the injector to the reversed phaseprimary column. The eluent post detection D1 flows to the sampling valveV1. In the home/analysis position, the primary column eluent flowsthrough the sampling loop Loop 1 exiting to waste. The mobile phase fromthe SFC pump flows through trapping column Trap 1 to the SFC column(FIG. 8A). This conditions the trapping column Trap 1 and SFC column.There is an uninterrupted flow of mobile phase through primary andsecondary columns. When components of interest elute from the primarycolumn, the valve V1 is switched (trapping position) transferring theprimary column eluent to the trapping column Trap 1 (FIG. 8B). Switchingthe valve V1 back to home/analysis position flushes the samplecomponents from trapping column T1 to the SFC column. The SFC columnseparation is monitored using UV detector D2 and/or a mass spectrometer.Interchanging positions of SFC pump and column in the valve V1 willresult in countercurrent flow during valve switching.

In 2D chromatography involving repetitive gradients in secondarydimension, the frequency of transferring (sampling) the primary columneluent into the secondary column depends on the resolving speed ofsecondary column including the re-equilibration time. In 2D LC-SFC, thesecondary dimension separation lasts about 2-3 minutes limiting thefrequency of transferring the primary column eluent to the secondarycolumn. If multiple analyte fractions (e.g., diastereomeric pairs in asample containing a mixture of stereoisomers) elute from the primarycolumn within 2-3 minutes, the 2D LC-SFC interface with a singletrapping column shown in FIG. 8A and FIG. 8B may not be practical.Slowing the primary column flow rate and gradient to make-up for thesampling needs of secondary column speed is an option. However, thiswill result in poor peak shape and hence preclude simultaneousachiral/chiral analysis. This will require an interface with multipletrapping columns to meet the sampling needs of the secondary column.

FIG. 9 shows schematics of an exemplary 2D LC-SFC system with aninterface using an array of trapping columns and secondary SFC columns.This configuration is used for analyzing multiple sections of thechromatogram. In the home/analysis position shown in FIG. 9, the eluentfrom the primary column post detection flows through a fully automated,2-position/4-port duo valve V1 exciting to waste. The SFC mobile phaseflows as follows: SFC pump through valve V1, to the parking deck valveV2, back to valve V1, and then to the SFC column(s). In valve V2, theSFC mobile phase flows either through by-pass tubing or an array oftrapping columns. This conditions the trapping and SFC column(s). Thereis an uninterrupted flow of mobile phase through primary and secondarycolumns. When components of interest elute from the primary column,valve V1 is switched (home to transfer) transferring the primary columneluent to parking deck valve V2 (Out). By switching the parking deckvalve V2 back and forth between the by-pass mode and trapping position,components are transferred to different trapping columns. Following thetransfer of the primary column eluent, valve V1 is switched to homeposition diverting the primary column eluent to waste. This reverses theflow of mobile phase coming into valve V2 (V2-Out to V2-In). Thedirection of SFC flow in the parking deck valve V2 is reversed.Components retained in the trapping columns are subsequentlyback-flushed into a SFC column or an array of SFC columns for furtherseparation depending upon the application. An array of SFC columnsprovides additional flexibility to the system as it might not bepractical to resolve different components on a single chiral stationaryphase. The secondary column separation is monitored using a UV or MSdetector. The automated interface is the key component of the 2D LC-SFCsystem enabling simultaneous achiral, chiral analysis of sample in asingle chromatographic run.

It is intended and understood that, in the 2D chromatograph system, eachand every variation of the first separation unit described herein may becombined with each and every variation of the second separation unitdescribed herein, and/or each and every combination of the first fluidicrouting unit described herein, as if each and every combination isindividually described. For example, in some embodiments, provided is a2D chromatograph system comprising:

(i) a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit;    -   c) a reversed-phase liquid chromatography (RPLC) column; and    -   d) a first detector;

(ii) a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit,    -   b) a supercritical fluid chromatography (SFC) column; and    -   c) a second detector;

and,

(iii) a first fluidic routing unit comprising a plurality of sampleloops, said first fluidic routing unit is connected to the firstseparation unit and the second separation unit,

-   -   wherein at least one of the plurality of sample loops comprises        a trapping column,    -   said trapping column comprising a stationary phase;

and

wherein the chromatography system is configured for first separating thesample in the first separation unit and subsequently introducing atleast a portion of the sample eluted from the RPLC column to the secondseparation unit.

In some embodiments, provided is a 2D chromatograph system comprising:

(i) a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit;    -   c) a reversed-phase liquid chromatography (RPLC) column; and    -   d) a first detector;

(ii) a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit,    -   b) a supercritical fluid chromatography (SFC) column; and    -   c) a second detector;

(iii) a first fluidic routing unit comprising two sample loops, saidfirst fluidic routing unit is connected to the first separation unit andthe second separation unit,

-   -   wherein one of the sample loops is in fluidic communication with        the first separation unit, and the other one of the sample loops        is in fluidic communication with the second separation unit;    -   and wherein at least one of the of sample loops comprises a        trapping column, said trapping column comprising a C-18        stationary phase (e.g., C-18 silica);

and,

(iv) at least one control device operably connected to one or more of:

-   -   a) the first pump assembly;    -   b) the sample injector;    -   c) the first detector;    -   d) the first fluidic routing unit;    -   e) the second pump assembly; and    -   f) the second detector;

and

wherein the chromatography system is configured for first separating thesample in the first separation unit and subsequently introducing atleast a portion of the sample eluted from the RPLC column to the secondseparation unit.

In one variation, the 2D chromatograph system further comprises at leastone control device operably connected to one or more of: a) the firstpump assembly; b) the sample injector; c) the first detector; d) thefirst fluidic routing unit; e) the second pump assembly; and f) thesecond detector.

In some embodiments, provided is a 2D chromatograph system comprising:

(i) a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit;    -   c) an RPLC column comprising a C-18 stationary phase (e.g., C-18        silica); and    -   d) a first detector;

(ii) a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit,    -   b) an SFC column comprising a normal phase stationary phase; and    -   c) a second detector;

(iii) a first fluidic routing unit comprising at least three sampleloops, said first fluidic routing unit is connected to the firstseparation unit and the second separation unit,

-   -   wherein one of the sample loops is in fluidic communication with        the first separation unit, another one of the sample loops is in        fluidic communication with the second separation unit, and at        least one of the sample loops is in fluidic isolation from the        first separation unit and the second separation unit;    -   and wherein at least one of the of sample loops comprises a        trapping column, said trapping column comprising a C-18        stationary phase (e.g., C-18 silica);

and,

(iv) at least one control device operably connected to one or more of:

-   -   a) the first pump assembly;    -   b) the sample injector;    -   c) the first detector;    -   d) the first fluidic routing unit;    -   e) the second pump assembly; and    -   f) the second detector;

and

wherein the chromatography system is configured for first separating thesample in the first separation unit and subsequently introducing atleast a portion of the sample eluted from the RPLC column to the secondseparation unit.

In one variation, the second separation unit comprises: a) an array ofSFC columns, wherein the SFC columns in the array are arranged in aparallel configuration; and b) a second fluidic routing unit fordirecting flow of the second mobile phase to a desired (orpre-identified) SFC column in the array. In another variation, thesecond separation unit further comprises a focus column (e.g., a focuscolumn comprising a C-18 stationary phase material (e.g., C-18 silica))positioned upstream of the SFC column.

In some embodiments, provided is a 2D chromatograph system comprising:

(i) a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit;    -   c) an RPLC column; and    -   d) a first detector;

(ii) a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit,    -   b) an array of SFC columns, wherein the SFC columns in the array        are arranged in a parallel configuration;    -   c) a second fluidic routing unit for directing flow of the        second mobile phase to a desired (or pre-identified) SFC column        in the array; and    -   c) a second detector;

and,

(iii) a first fluidic routing unit comprising at least three sampleloops, said first fluidic

routing unit is connected to the first separation unit and the secondseparation unit,

-   -   wherein one of the sample loops is in fluidic communication with        the first separation unit, another one of the sample loops is in        fluidic communication with the second separation unit, and at        least one of the sample loops is in fluidic isolation from the        first separation unit and the second separation unit;    -   and wherein at least one of the of sample loops comprises a        trapping column, said trapping column comprising a stationary        phase;

and

wherein the chromatography system is configured for first separating thesample in the first separation unit and subsequently introducing atleast a portion of the sample eluted from the RPLC column to the secondseparation unit.

In some variations, the RPLC column comprises a C-18 stationary phase(e.g., C-18 silica). In some variations, the SFC column comprises anormal phase silica stationary phase. In some variations, the systemfurther comprises at least one control device operably connected to oneor more of:

a) the first pump assembly;

b) the sample injector;

c) the first detector;

d) the first fluidic routing unit;

e) the second pump assembly; and

f) the second detector.

The 2D RPLC×SFC system of the present invention address the problemassociated with the incompatibility of solvents used in the firstdimension RPLC and the second dimension SFC by using a trapping column.The stationary phase in the trapping column retains the analytes whileletting the solvents from the RPLC to flow through. This allows theanalytes to be concentrated in a small volume for injection into the SFCfor further separation. A higher water content in the SFC dimensionleads to lower resolution/sensitivity. As demonstrated in Example 2,using a system without a trapping column, only a small fraction can betransferred to the SFC column without adversely affecting the resolutionand sensitivity of the SFC analysis (FIG. 11). A transfer volume of 12μL led to mild broadening of the second peak. When a 24 μL fraction wastransferred, a significant broadening of the second peak is observed,which translates to significant loss of sensitivity. In contrast, asshown in Example 3, when a system as depicted in FIGS. 8A and 8B wasused, which has a trapping column containing a C-18 stationary phasematerial (e.g., C-18 silica), a window of 160 μL was transferred, andexcellent resolution and sensitivity were obtained (FIG. 12). Thissystem allows for injection of peaks from the first dimension RPLC tothe second dimension SFC with minimal impact on the resolution andsensitivity of the SFC analysis. For example, Example 5 comparesseparation of a chiral drug substance with its enantiomer using aconventional SFC system and a 2D LC-SFC system of the present invention.The results demonstrated that both resolution and sensitivity werepreserved in the 2D LC-SFC as compared to conventional SFC. Theorthogonal approach, reversed-phase and normal phase conditions in thetwo dimensions, can be used to increase the confidence level of HPLCpeak purity assessment due to the multiplicative peak capacity of themultidimensional system.

Methods of Use

Another factor to consider when developing 2D systems is the ability tooperate in an on-line mode. Some advantages of this approach include theease of automation, reproducibility of the analysis, and the accuratetransfer of the fractions from the first to the second dimension withoutany yield loss or contamination.

An overlooked application of 2D systems is the use in high-throughputanalysis. In the pharmaceutical industry for example, ActivePharmaceutical Ingredients (APIs) have to be fully characterized per ICHguidelines. See International Conference on Harmonisation (2006),Q3A(R2): Impurities in New Drug Substances. For purity analysis, twoindependent analytical methods are developed. A RPLC method usuallyassesses the achiral purity (impurities and related substances method),and a chiral method that would assess the chiral purity (amount ofundesired enantiomer). A 2D system that can generate simultaneousachiral and chiral results would have a huge impact during API processdevelopment. Sample preparation, chromatographic analysis times, anddata analysis would be reduced to allow higher throughput analysis.

We have previously reported the use of 2D RPLC×RPLC analysis forsimultaneous achiral-chiral analysis (J. Sep. Sci. 2012, 35:1748). Inthe API world however, the majority of chiral methods are NPLC methods,and thus a 2D RPLC×NPLC system would have a significant bearing inachieving simultaneous achiral-chiral analysis. As noted above, theincompatibility of the reversed phase and normal phase mobile phaseswould make this approach very challenging. Supercritical fluidchromatography, a normal phase technique, has also been used for APIchiral analysis on analytical as well as preparative scale. In additionto being a “green” technique, SFC is superior to NPLC due to itsversatility, higher efficiency, higher throughput, and faster analysistimes. Supercritical fluids have low viscosity and high diffusivity(similar to gases) to allow higher flow rates and fasterre-equilibration times and have a high density (similar to liquids) toprovide a high solvating power. The first on-line 2D LC×SFC was reportedby Cortes et al. in 1992 (J. Microcol. September. 1992, 4:239-244). Theinterface that Cortes et al. developed is rather complicated andinvolves multiple stages: elimination of the first dimension solvent bythe passage of nitrogen gas, using pressurized CO₂ to transfer theanalytes onto an impactor interface, and then elution of the analytesfrom the impactor interface to the SFC capillary column by pressureprogramming of the CO₂ mobile phase. The adoption of this interface forconventional 2D RPLC×SFC separations would be limited due to the solventelimination step. Cortes et al. used THF (relatively low boiling point,66° C.) as the LC mobile phase while most conventional RPLC separationsare aqueous based.

The present invention demonstrates a new automated interface to coupleRPLC and SFC. Thus provided are methods of using the 2D RPLC×SFCchromatography systems described herein for separation and analysis ofsamples, for example complex sample mixtures which may be difficult toachieve comprehensive analysis by 1D chromatography or other 2Dchromatography.

In some embodiments, provided is a method for analyzing a sample (suchas a complex sample) using a chromatography system described hereincomprising: first separating the complex sample into fractions byreversed-phase liquid chromatography (RPLC); and further separating thefractions from the RPLC dimension by supercritical fluid chromatography(SFC) in the second dimension. Separation in the first dimension (e.g.,RPLC on a C-18 stationary phase) relies on differences of certaincharacters or properties of the components in the complex sample (e.g.,hydrophobicity); while separation in the second dimension (e.g., SFC ona normal phase silica gel stationary phase) relies on differences ofother characters or properties of the components (e.g., chirality), thusproviding better comprehensive analysis than using one-dimensionalchromatography.

Developing chiral chromatographic methods for compounds with multiplechiral centers can be challenging as the number of potentialstereoisomers increases significantly with the increase in number of thechiral centers (Number of stereoisomers=2^(N), where N is the number ofchiral centers in a compound). Chiral method development is mostly atrial and error process where extensive column and mobile phasescreening is done to identify potential hits. However, developing chiralchromatographic methods for compounds with 3 or more chiral centers canbe very challenging due to the significant increase in the number ofstereoisomers. For pharmaceutical compounds with multiple chiralcenters, one common practice is to control the enantiomeric purity ofincoming starting materials and demonstrate process control (possibilityof epimerization). This would limit the number of potentialstereoisomers in the final API. However, this control strategy could bechallenged by some regulatory agencies mandating the development of anAPI chiral method.

The online 2D RPLC×SFC of the present invention allows simultaneousachiral and chiral analysis (analysis by a single sample injection or inthe same analytical run) of pharmaceutical samples. The API peak, in amixture of aqueous and organic content, would be retained on a smallvolume C-18 trapping column and then back-flushed onto the seconddimension SFC column. Thus, in some embodiments, provided is a methodfor achiral-chiral analysis of a sample comprising a mixture ofstereoisomeric components using a chromatography system described hereincomprising: resolving diastereomeric components in the sample by RPLC inthe first dimension, which provides achiral purity of the sample; andresolving the enantiomeric pairs by SFC in the second dimension in thesame analytical run, which further provides chiral purity (%enantiomeric excess) of the components in the sample. The achiral purityof the sample may be determined based on a chromatogram from the RPLCseparation, for example, by relative peak area of peaks on achromatogram obtained on a UV detector of the first separation unit. Thechiral purity or enantiomeric excess of each enantiomeric pair may bedetermined based on a chromatogram from the SFC separation, for example,by relative peak area of peaks on a chromatogram obtained on a UVdetector of the second separation unit, or total ion chromatogramobtained on a MS spectrometer attached to the second separation unit.

It is intended and understood that each and every embodiments of the 2Dchromatograph system may be used in the methods for analyzing a complexsample or the methods for simultaneous achiral-chiral analysis as ifeach and every combination is individually described. For example, insome embodiments, provided is a method for simultaneous achiral-chiralanalysis of a sample comprising a mixture of stereoisomeric componentsusing a 2D chromatography system, said 2D chromatography systemcomprising:

(i) a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit;    -   c) a reversed-phase liquid chromatography (RPLC) column; and    -   d) a first detector;

(ii) a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit,    -   b) a supercritical fluid chromatography (SFC) column; and    -   c) a second detector;

and,

(iii) a first fluidic routing unit comprising a plurality of sampleloops, said first fluidic routing unit is connected to the firstseparation unit and the second separation unit,

-   -   wherein at least one of the plurality of sample loops comprises        a trapping column, said trapping column comprising a stationary        phase;

said method comprising: first resolving diastereomeric components in thesample by RPLC on the first separation unit, and then resolving theenantiomeric pairs by SFC on the second separation unit in the sameanalytical run.

In some embodiments, provided is a method for simultaneousachiral-chiral analysis of a sample comprising a mixture ofstereoisomeric components using a 2D chromatography system, said 2Dchromatography system comprising:

(i) a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit;    -   c) an RPLC column; and    -   d) a first detector;

(ii) a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit,    -   b) an array of SFC columns, wherein the SFC columns in the array        are arranged in a parallel configuration;    -   c) a second fluidic routing unit for directing flow of the        second mobile phase to a desired (or pre-identified) SFC column        in the array; and    -   d) a second detector;

and,

(iii) a first fluidic routing unit comprising at least three sampleloops, said first fluidic routing unit is connected to the firstseparation unit and the second separation unit,

-   -   wherein one of the sample loops is in fluidic communication with        the first separation unit, another one of the sample loops is in        fluidic communication with the second separation unit, and at        least one of the sample loops is in fluidic isolation from the        first separation unit and the second separation unit;    -   and wherein at least one of the of sample loops comprises a        trapping column, said trapping column comprising a stationary        phase;        said method comprising: first resolving diastereomeric        components in the sample by RPLC on the first separation unit,        and then resolving the enantiomeric pairs by SFC on the second        separation unit in the same analytical run.

In some embodiments, provided is a method for simultaneousachiral-chiral analysis of a sample comprising a mixture ofstereoisomeric components using a 2D chromatography system, said 2Dchromatography system comprising:

(i) a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit;    -   c) an RPLC column; and    -   d) a first detector;

(ii) a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit,    -   b) an SFC column; and    -   c) a second detector;

(iii) a first fluidic routing unit comprising at least three sampleloops, said first fluidic routing unit is connected to the firstseparation unit and the second separation unit,

-   -   wherein one of the sample loops is in fluidic communication with        the first separation unit, another one of the sample loops is in        fluidic communication with the second separation unit, and at        least one of the sample loops is in fluidic isolation from the        first separation unit and the second separation unit;    -   and wherein at least one of the of sample loops comprises a        trapping column, said trapping column comprising a stationary        phase;

and,

(iv) at least one control device operably connected to one or more of:

-   -   a) the first pump assembly;    -   b) the sample injector;    -   c) the first detector;    -   d) the first fluidic routing unit;    -   e) the second pump assembly; and    -   f) the second detector;        said method comprising: first resolving diastereomeric        components in the sample by RPLC on the first separation unit,        and then resolving the enantiomeric pairs by SFC on the second        separation unit in the same analytical run.        Methods

In another aspect, provided is a method for separating a sample bymulti-dimensional chromatography (such as 2D RPLC×SFC) comprisingsubjecting a portion of a sample captured on a trapping column, saidportion is obtained by separating the sample by reversed-phase liquidchromatography (RPLC), said trapping column comprising a stationaryphase, to further separation by supercritical fluid chromatography(SFC). In some embodiments, provided is a method for separating a samplecomprising the steps of: (i) capturing at least a portion of a sample ona trapping column, said portion is obtained by separating the sample byreversed-phase liquid chromatography (RPLC), said trapping columncomprising a stationary phase; and (ii) subjecting the portion of thesample captured on the trapping column to further separation bysupercritical fluid chromatography (SFC).

In some embodiments, the method further comprises a step of separatingthe sample by reversed-phase liquid chromatography (RPLC), comprising:(i) introducing the sample into a first mobile phase; (ii) driving thefirst mobile phase containing the sample through a RPLC column; and(iii) separating the sample on the RPLC column. In some embodiments, themethod further comprises detecting the presence of a component of thesample in the first mobile phase after passing through the RPLC column.In some embodiments, the method further comprises eluting the portion ofthe sample captured on the trapping column off the trapping column. Insome embodiments, the method further comprises detecting a component ofthe sample after further separation by SFC.

In some embodiments, the method further comprises positioning a trappingcolumn in a flow path of the first mobile phase downstream of the RPLCcolumn for capturing at least a portion of the sample separated by theRPLC column, and/or switching the trapping column carrying the capturedportion to a flow path of a second mobile phase for eluting the capturedportion off the trapping column. The positioning/switching of thetrapping column in/out of the flow path of the mobile phase of theRPLC/SFC unit may be performed in a fluidic routing device interfacingthe RPLC unit and the SFC unit in a 2D RPLC×SFC chromatography system.

In some embodiments, the method may be performed on a 2D RPLC×SFCchromatography system configured for countercurrent elution of theanalytes captured on the trapping column. In such variation, the firstmobile phase flows through the trapping column in a first direction, andthe portion of the sample captured on the trapping column is eluted offthe trapping column by flowing the second mobile phase through thetrapping column in a direction opposite to the first direction. In someembodiments, the method may be performed on a 2D RPLC×SFC chromatographysystem configured for co-current elution of the analytes captured on thetrapping column. In such variation, the first mobile phase flows throughthe trapping column in a first direction, and the portion of the samplecaptured on the trapping column is eluted off the trapping column byflowing the second mobile phase through the trapping column in the samedirection as the first direction.

In some embodiments, a method for separating a sample with amulti-dimensional chromatography (e.g., a 2D RPLC×SFC chromatographysystem described herein, or any variations thereof) is provided,comprising the steps of:

-   -   (i) introducing a sample into a first mobile phase;    -   (ii) driving the first mobile phase containing the sample        through a RPLC column;    -   (iii) separating the sample on the RPLC column;    -   (iv) detecting the presence of a component of the sample in the        first mobile phase after passing through the RPLC column;    -   (v) capturing on a first trapping column at least a first        portion of the sample separated on the RPLC column, said first        trapping column comprising a stationary phase;    -   (vi) eluting the first portion of the sample captured on the        first trapping column off the first trapping column;    -   (vii) subjecting the first portion of the sample captured on the        first trapping column to further separation by SFC; and    -   (viii) detecting a component of the sample after further        separation by SFC.

In some instances of 2D chromatography, such as in the comprehensive orpseudo-comprehensive mode, more than one fractions from the firstdimension RPLC may be captured one or more trapping columns and releasedfor analysis in the second dimension SFC. Thus in some embodiments, themethod further comprises the steps of:

-   -   (ix) capturing on a second trapping column at least a second        portion of the sample separated on the RPLC column, said second        trapping column comprising a stationary phase;    -   (x) eluting the second portion of the sample captured on the        second trapping column off the second trapping column;    -   (xi) subjecting the second portion of the sample captured on the        second trapping column to further separation by SFC.        These steps may be repeated multiple times for        capturing/releasing multiple fractions.

In some embodiments, provided is a method for separating a sample usinga multi-dimensional chromatography (e.g., a 2D RPLC×SFC chromatographysystem described herein, or any variations thereof), comprising thesteps of:

-   -   (i) introducing a sample into a first mobile phase;    -   (ii) driving the first mobile phase containing the sample        through a RPLC column;    -   (iii) separating the sample on the RPLC column;    -   (iv) detecting the presence of a component of the sample in the        first mobile phase after passing through the RPLC column;    -   (v) positioning a trapping column in a flow path of the first        mobile phase downstream of the RPLC column;    -   (vi) capturing on the trapping column at least a portion of the        sample separated on the RPLC column, said trapping column        comprising a stationary phase;    -   (vii) switching the trapping column carrying the captured        portion into a flow path of a second mobile phase;    -   (viii) eluting the portion of the sample captured on the        trapping column off the trapping column;    -   (ix) subjecting the portion of the sample captured on the        trapping column to further separation by SFC; and    -   (x) detecting a component of the sample after further separation        by SFC.        In some embodiments, steps (i) through (x) are performed in the        order as listed. In some embodiments, the steps (iv) through (x)        are repeated for one or more times until all fractions of        interests are analyzed.

In some of these embodiments, the RPLC column comprises a reversed-phasestationary phase, for example, a stationary phase comprising areversed-phase material such as a C-18 phase (e.g., a C-18 silica), aC-8 phase (e.g., a C-8 silica), a C-4 phase (e.g., a C-4 silica), orother reversed-phase materials described herein. In some of theseembodiments, the stationary phase in the trapping column comprises areversed-phase material such as a C-18 phase (e.g., a C-18 silica), aC-8 phase (e.g., a C-8 silica), a C-4 phase (e.g., a C-4 silica), orother reversed-phase materials described herein.

In some of these embodiments, the SFC separation is performed on a SFCcolumn comprising a normal phase stationary phase, for example, astationary phase comprising a normal phase silica gel or other normalphase materials described herein. In some of these embodiments, the SFCseparation is performed on a SFC column selected from an array of SFCcolumns arranged in parallel, each SFC column in the array may comprisea stationary phase that may be the same or different. The stationaryphase material in the SCF column may be adapted for separating thespecific components in the sample.

In some of these embodiments, the method may further comprise focusingthe analytes eluted from the fraction captured on the trapping column ona focus column prior to further separation on the SFC column. The focuscolumn comprises a stationary phase that may or may not be the same asthe stationary phase used in the trapping column. In some embodiments,the focus column is loaded with a stationary phase comprising areversed-phase material such as a C-18 phase, a C-8 phase, a C-4 phaseor other reversed-phase materials described herein.

The methods for separating a sample described herein may be adapted forperformance on a 2D RPLC×SFC chromatography system described herein orany embodiments or variations thereof described.

EXAMPLES

The following Examples are provided to illustrate but not to limit theinvention.

Chemicals and Regents

Carbon dioxide (CO₂) was obtained from Praxair (Danbury, Conn., USA).Acetonitrile (ACN) was purchased from Avantor's J. T. Baker (CenterValley, Pa., USA). Methanol (MeOH), isopropyl alcohol (IPA), ethylalcohol (EtOH), 98.0%-100.0% formic acid, and 28.0%-30.0% ammoniumhydroxide (NH₄OH) were purchased from EMD chemicals (Gibbstown, N.J.,USA). Ammonium formate was purchased from Sigma Aldrich (St. Louis, Mo.,USA). HPLC grade Millipore water was obtained from Purelab ultraMillipore water dispenser. Trans-stilbene oxide (TSO) was purchased fromTCI (Tokyo, Japan). Drug substance A used in this study was synthesizedby the process chemistry department at Genentech, CA, USA.

Example 1 Instrumentation

The analytical instrument is a customized two-dimensional 1260 2D-LC-SFCsystem with mass spectrometer from Agilent Technologies (Santa Clara,Calif., USA). The RPLC unit consists of an Agilent 1260 quaternary pump(G1311B), a 1260 HiP ALS auto-sampler (G1367E), and an Agilent 1260multi-wavelength UV detector (G1365C). Stainless steel fittings andtubing are used throughout the system due to high pressureconsiderations. The SFC unit consists of a 1260 SFC binary pump (G4302A)with a three position solvent control valve, a 1260 HiP degasser(G4225A), a 1290 thermostated column compartment (G1316C), aneight-position Agilent 1290 infinity valve drive (G1170A), an Agilent1260 DAD (G1315C) equipped with a high-pressure flow cell, and anAgilent 1260 infinity SFC control module (G4301A). Part of the SFC flowwas directed towards an Agilent 6120 quadrupole MS. An Agilent 1260 isopump (G1310B) was used to generate a make-up flow of 0.15 mL/min inorder to compensate for the loss of scCO₂. An Agilent 1290 Flexcube(G4227A) was installed to enable multiple peak parking on differenttrapping columns using a custom built 12-port switching valve.Instrument control and data collection was done with Agilent Chemstationsoftware (Santa Clara, Calif., USA).

FIG. 10 is a photographic image of an exemplary parking deck valve(e.g., valve V2 featured in FIG. 9). As shown in FIG. 10, there are fourtrapping columns that may be used for peak parking. Additionally, asdepicted, there is a by-pass loop that allows for either the mobilephase originating from the first separation unit or the mobile phaseoriginating from the second separation unit to be directed downstreamwithout passing through a trapping column.

Example 2

The primary objective of this study was to evaluate the effect of thevolume of the mobile phase transferred from the first dimension to thesecond dimension on the resolution of the separation in the seconddimension (SFC). Here, a multidimensional chromatography system was usedto separate the enantiomers of trans-stilbene oxide (TSO). The firstdimension (RPLC) was interfaced with the second dimension (SFC) using avalve with a sample loop lacking a trapping column. The sample loop wasused to store a selected volume of the first mobile phase containing aportion of the sample eluted from the first dimension. Subsequently, thevolume of the first mobile phase stored in the sample loop wastransferred to the second dimension for further separation. Sample loopsallowing for the storage, and subsequent transfer, of 6 μL, 12 μL, and24 μL of mobile phase were used.

For the first RPLC dimension, an ACQUITY UPLC HSS T3 1.8 μm 2.1×50 mmcolumn was utilized under an isocratic condition with 90/10 ACN/water ata flow rate of 0.2 mL/min. UV detection was done at 225 nm.

For the second SFC dimension, a Chiralcel OD3 3.0 μm 4.6 mm×50 mm columnwas utilized at 40° C. with an isocratic flow of 95:5 scCO₂ (MPA)/IPAwith 0.1% NH4OH (MPB). The flow rate was 4.0 mL/min with an outletpressure of 130 bar and nozzle temperature of 60° C.

The trapping loop for this experiment was a flex capillary (0.5 mm×150mm with an internal volume of approximately 29 μL). The three switchingtimes were 0.03 min, 0.06 min, and 0.12 min corresponding to 6 μL, 12μL, and 24 μL transfer volumes, respectively (based on a flow rate of0.2 mL/min).

FIG. 11 is a compilation chromatograph of the UV absorbance measurements(mAU) over time (minutes) for the multidimensional separation of TSOusing a system with three sample loop configurations (6 μL, 12 μL, 24μL). As illustrated in FIG. 11, increasing the transfer volume of thefirst mobile phase to the second dimension reduces the resolution andsensitivity in the second dimension.

Example 3

In this example, the efficiency of transferring a sample from a firstdimension (RPLC) to a second dimension (SFC) using an interfacecontaining a trapping column was evaluated.

Solutions of TSO standard ranging from 0.005 mg/mL to 0.25 mg/ml wereanalyzed using the 2D LC-SFC system illustrated in FIG. 8A and FIG. 8B.The second detector was a UV detector. Based on the results from Example2, a C18-reversed-phase trapping column of low internal volume wasevaluated.

The reversed-phase chromatograph utilized in the first dimension was anAcquity UPLC HSS T3 column (50×2.1 mm, 1.8 μm) from Waters Corporation(Milford, Mass., USA). The separation in the first dimension was rununder isocratic conditions with 50:50 (0.05% formic acid inwater):(0.05% formic acid in ACN) at a flow rate of 0.2 mL/min. The RPLCcolumn was placed in the SFC thermal column compartment at 40° C. UVdetection was done at 225 nm. The first dimension injection volume was 5μL.

The supercritical fluid chromatograph used in the second dimension was aChiralcel OD3 column (50×4.6 mm, 3.0 μm) from Chiral Technologies (WestChester, Pa., USA). The separation in the second dimension was run underan isocratic flow of 95:5 (scCO2):(isopropyl alcohol with 0.1% ammoniumhydroxide). The column temperature used was 40° C. and the flow rate wasset at 4.0 mL/min with an outlet pressure of 130 bar and nozzletemperature of 60° C. Using the focusing column at the head of SFCcolumn is optional.

As discussed above, a low volume trapping column was used in theinterface of the two dimensions. Specifically, the trapping column usedwas a SunShell C18 column (5.0×1.0 mm, 5 μm) from ChromaNik Technologies(Osaka, Japan).

TSO standard solutions (0.25, 0.1, 0.05, 0.025, 0.01, 0.005 mg/mL) wereprepared in 50:50 ACN/ water. A window of 0.8 min (˜160 μL) across theapex of the TSO peak was transferred to the trapping column that wasconditioned with initial SFC conditions (100% scCO₂). An initial hold at0% (IPA with 0.1% NH4OH) was maintained for first 0.2 min after theswitch and then increased to 5% (IPA with 0.1% NH4OH) in 0.1 min with a2.35 min hold. The column was re-equilibrated with 0% (IPA with 0.1%NH4OH) for 0.2 min. Samples were run in triplicates. Detection in thesecond dimension was done by UV detection at 225 nm.

FIG. 12 is a compilation chromatograph of the UV absorbance measurements(mAU) over time (minutes) for the multidimensional separation of varyingconcentrations of TSO. The top chromatogram was measured followingseparation in the first dimension. Enantiomers of TSO standard areobserved to co-elute in the reverse phase primary column. The peakeluting from the primary column post detection was diverted to thetrapping column and back-flushed into the secondary column for furtherseparation. As shown in FIG. 12, the bottom chromatograph was measuredfollowing separation in the second dimension. Here, enantiomers of TSOstandard are observed to be baseline resolved in the secondary chiralcolumn.

Furthermore, as illustrated in FIG. 12, overlay plots demonstrated thelinearity of detector response over the evaluated concentration rangewith a correlation coefficient greater than 0.99 (data not shown). In adifferent study, volumes ranging from 10 to 100 μL (by timing valve V1)were transferred to the secondary SFC column. The results of this studyshowed linear response over the evaluated range (results not shown).

Example 4

In this example, the 2D LC-SFC system described in Example 3 was furthertested to demonstrate the ability of simultaneous achiral-chiralanalysis of a sample of Drug Substance A.

The reversed-phase chromatogram utilized in the first dimension was aSunFire C18 column (150×3.0 mm, 3.5 μm) from Waters Corporation(Milford, Mass., USA) at a temperature of 40° C. MPA was 5 mM ammoniumformate, pH 3.3 and MPB was 0.05% formic acid in ACN. MP program for theRPLC column was 5% B to 25% B in 5 min, to 29% B in 25 min, to 90% B in30 min, and then re-equilibration at 5% B for 5 min. The flow rate wasset to 1.0 mL/min. The first dimension UV detection was done at 340 nm.The first dimension injection volume was 5 μL.

The supercritical fluid chromatogram used in the second dimension was aChiralpak IC3 column (50×4.6 mm, 3 μm) from Chiral Technologies (WestChester, Pa., USA) at 40° C. with an initial MP flow of 65:35 scCO₂(MPA)/methanol with 0.1% ammonium hydroxide (MPB). The flow rate was 4.0mL/min with an outlet pressure of 130 bar and nozzle temperature of 60°C. Four Zorbax Eclipse XDB-C18 columns (5.0×2.1 mm, 1.8 μm) from AgilentTechnologies (Santa Clara, Calif., USA) were used as the trappingcolumn.

A sample of Drug Substance A was prepared at 0.5 mg/mL in 25:75ACN/water with 0.05% FA. A window of 0.1 min (100 μL) across the apexwas transferred to the pre-conditioned trapping column. The SFC columnwas maintained at an isocratic hold (35% MPB) for 0.5 min, then to 55% Bin 2 min with a 3 min hold. The column was re-equilibrated at 35% B for0.2 min. Detection in the second dimension was done by SIM-MS detectionat 565 m/z.

As depicted in FIG. 13, the achiral and chiral purity results were 99.0%and 100% enantiomeric excess (% ee), respectively. An overlay plot ofunspiked sample with no enantiomer detected (bottom), sample spiked with0.1% of the undesired enantiomer (middle), and a sample spiked with 0.5%undesired enantiomer (top) demonstrated the capability of this system todetect the undesired enantiomer at 0.1% levels.

Example 5

The present study was performed to demonstrate the comparability of bothsensitivity and resolution between the second dimension SFC in a 2DLC-SFC system and conventional (1D) SFC.

The 2D LC-SFC system was the same as the system used in Example 4. Themobile phase program and switching time were modified. In the firstdimension, the mobile phase program was 25% B for 5 min, 25% to 90% B by15 min, and then re-equilibrated at 25% B for 5 min. A window of 0.1 min(100 μL) at the apex of the peak was transferred to the trapping column.The conditions for conventional SFC were the same as those used in thesecond dimension of 2D LC-SFC (described in Example 4).

Standard solutions of a sample of Drug Substance A containing varyinglevels of the undesired enantiomer (0.1 to 2.0% range) were analysedusing both techniques (2D LC-SFC and SFC). An overlay of the standardchromatograms from SFC and 2D LC-SFC techniques are shown in FIG. 14Aand FIG. 14B, respectively. The results illustrated comparableseparations obtained with both techniques. Both resolution andsensitivity were preserved in the 2D LC-SFC as compared to conventionalSFC.

As demonstrated in Example 2, introduction of reversed-phase mobilephase into the SFC dimension deleteriously affects resolution andsensitivity of SFC separation. Although reversed-phase mobile phase isstill introduced into the SFC dimension with the 2D LC-SFC systemdescribed here, use of a trapping column allows for an LC-SFC interfacethat does not compromise downstream SFC separation.

Example 6

In this example, a complex chiral chromatographic separation of desiredsensitivity and selectivity in a single analysis is demonstrated.

Drug Substance A has three chiral centers and hence has four pairs ofdiastereomers (eight potential stereoisomers). A mixture of the 4diastereomeric pairs was prepared in 30/70 ACN/water at 0.05 mg/mL.Ratio of the two enantiomers in each pair (RRS/SRR, SRS/RSR, SSS/RRR;RRS/SSR) was approximately 2:1.

Separation of each stereoisomer was achieved using the 2D LC-SFC systemshown in FIG. 9. The experimental conditions in the primary column weresame as one described in section Example 4. Four trapping columns wereused with the Flexcube (Agilent) in the secondary dimension to trap the4 diastereomeric pairs. Following sample injection, trapping columnswere conditioned with 60:40 scCO₂ (MPA)/methanol with 0.1% ammoniumhydroxide (MPB) for one min with the exception of trapping column 2which was conditioned with 65:35 scCO₂ (MPA)/methanol with 0.1% ammoniumhydroxide (MPB). Primary column eluent corresponding to 0.1 min (100 μL)window at the apex of the diastereomeric peaks at 10.55 min, 10.95 min,11.80 min and 13.30 min were sequentially transferred into four trappingcolumns. Trapped components were sequentially chromatographed startingat 14.0 min, 18.0 min, 23.5 min and 27.5 min respectively in thesecondary dimension. An initial hold at 60:40 scCO₂ (MPA)/methanol with0.1% ammonium hydroxide (MPB) was maintained for first 0.5 min after theswitch and then increased to 40:60 scCO₂ (MPA)/methanol with 0.1%ammonium hydroxide (MPB) in 2.5 min with a 0.3 min hold with theexception of trapped component 2. For component 2, the initial hold at65:35 scCO₂ (MPA)/methanol with 0.1% ammonium hydroxide (MPB) wasmaintained for first 1 min after the switch and then increased to 55:45scCO₂ (MPA)/methanol with 0.1% ammonium hydroxide (MPB) in 3.0 min witha 0.3 min hold. Detection in the second dimension was done by SIM-MSdetection at 565 m/z.

As illustrated in FIG. 15, the primary achiral RPLC column resolves thefour diastereomeric pairs (RSS/SRR, SRS/RSR, RRR/SSS, SSR/RRS) and otherprocess related impurities from the API providing achiral purity. Eachof these diastereomeric pair is then sequentially transferred from theprimary RPLC column (post detection) to four different trapping columnsin Valve 2 (V2; FIG. 9). The trapped diastereomeric fractions are thensequentially back-flushed and analysed on the secondary SFC chiralcolumn providing chiral purity. By presenting a simpler sample mixtureto the secondary chiral column, potential stereoisomers are moreefficiently resolved. As shown in FIG. 15, eight stereoisomers,corresponding to the four-diastereomeric pairs, were successfullyresolved on the secondary SFC dimension using MS detection. Using theparking deck valve, the application of the 2D LC-SFC is extended to theanalysis of compounds with multiple chiral centers that are difficult toresolve by conventional chiral chromatography.

Example 7

The present study was performed to test a 2D LC-SFC system wherein afocus column is placed at the head of the SFC column.

The 2D LC-SFC conditions were the same as those described in Example 3.Furthermore, the TSO samples were prepared in the same manner asdescribed in Example 3. In short, TSO standard solutions (0.25, 0.1,0.05, 0.025, 0.01, 0.005 mg/mL) were prepared in 50:50 ACN/water. Thefirst dimension injection volume was 5 μL. In addition, for thoseanalyses complete with a focusing column, a focusing column was placedat the head of the SFC column. The focusing column used was Pursuit XRsC18 (20×2.0 mm, 5 um) from Agilent Technologies (Santa Clara, Calif.,USA).

The 2D LC-SFC separation of a series of concentrations of a TSO standardwith and without a focusing column is shown in FIG. 16. The TSOenantiomers are baseline resolved in both conditions. Using a focusingcolumn however, resulted in increasing the slope of peak height v/s peakarea plot improving the signal to noise (S/N) ratio in the seconddimension (peak 1). Similar results were observed for peak 2 (data notshown).

Exemplary Embodiments

The invention is further described by the following embodiments. Thefeatures of each of the embodiments are combinable with any of the otherembodiments where appropriate and practical.

Embodiment 1. In one embodiment, the invention provides a chromatographysystem for separating a sample comprising:

a first separation unit comprising:

-   -   a) a first pump assembly for driving a first mobile phase        through the first separation unit,    -   b) a sample injector for introducing a sample to the first        separation unit; and    -   c) a reversed-phase liquid chromatography (RPLC) column;

a second separation unit comprising:

-   -   a) a second pump assembly for driving a second mobile phase        through the second separation unit, and    -   b) a supercritical fluid chromatography (SFC) column;

and,

a first fluidic routing unit comprising a plurality of sample loops,said first fluidic routing unit is connected to the first separationunit and the second separation unit,

-   -   wherein at least one of the plurality of sample loops comprises        a trapping column,    -   said trapping column comprising a stationary phase;

and

wherein the chromatography system is configured for first separating thesample in the first separation unit and subsequently introducing atleast a portion of the sample eluted from the RPLC column to the secondseparation unit.

Embodiment 2. In a further embodiment of embodiment 1, the first fluidicrouting unit comprises two sample loops; wherein one of the two sampleloops is in fluidic communication with the first separation unit and theother one of the two sample loops is in fluidic communication with thesecond separation unit.

Embodiment 3. In a further embodiment of embodiment 1, wherein the firstfluidic routing unit comprises at least three sample loops, and whereinat least one of the sample loops is in fluidic isolation from the firstseparation unit and the second separation unit.

Embodiment 4. In a further embodiment of embodiment 3, at least onesample loop comprising a stationary phase material is in fluidicisolation from the first separation unit and the second separation unit.

Embodiment 5. In a further embodiment of embodiment 1, the first fluidicrouting unit comprises a plurality of trapping columns each positionedin a sample loop.

Embodiment 6. In a further embodiment of any one of embodiments 1 to 5,the first fluidic routing unit is configured to allow fluid flow througha sample loop in a first direction when said sample loop is positionedin fluidic communication with the first separation unit and to allowfluid flow through said sample loop in a direction opposite to the firstdirection when said sample loop is positioned in fluidic communicationwith the second separation unit.

Embodiment 7. In a further embodiment of any one of embodiments 1 to 5,the first fluidic routing unit is configured to allow fluid flow througha sample loop in a first direction when said sample loop is positionedin fluidic communication with the first separation unit and to allowfluid flow through said sample loop in a direction same as the firstdirection when said sample loop is positioned in fluidic communicationwith the second separation unit.

Embodiment 8. In a further embodiment of any one of embodiments 1 to 7,the RPLC column comprises a reversed-phase stationary phase.

Embodiment 9. In a further embodiment of embodiment 8, thereversed-phase stationary phase comprises a C-18 phase (e.g., C-18silica).

Embodiment 10. In a further embodiment of embodiment 8 or 9, thestationary phase in the trapping column comprises a reversed-phasematerial.

Embodiment 11. In a further embodiment of embodiment 9, thereversed-phase material comprises a C-18 phase (e.g., C-18 silica).

Embodiment 12. In a further embodiment of any one of embodiments 1 to11, the second separation unit comprises one SFC column.

Embodiment 13. In a further embodiment of embodiment 12, the SFC columncomprises a normal phase stationary phase.

Embodiment 14. In a further embodiment of embodiment 13, the normalphase stationary phase comprises a silica gel.

Embodiment 15. In a further embodiment of any one of embodiments 1 to14, the second separation unit further comprises a focus column locatedupstream of the SFC column.

Embodiment 16. In a further embodiment of embodiment 15, the focuscolumn comprises a reversed-phase material.

Embodiment 17. In a further embodiment of any one of embodiments 1 to11, the second separation unit comprises: a) an array of SFC columns,wherein the SFC columns in the array are arranged in a parallelconfiguration; and b) a second fluidic routing unit for directing flowof the second mobile phase to a desired (or pre-identified) SFC columnin the array.

Embodiment 18. In a further embodiment of embodiment 17, the secondseparation unit further comprises a focus column located upstream ofeach SFC column in the array of SFC columns.

Embodiment 19. In a further embodiment of any one of embodiments 1 to18, further comprising a first detector positioned downstream of theRPLC column.

Embodiment 20. In a further embodiment of any one of embodiments 1 to19, further comprising a second detector positioned downstream of theSFC column.

Embodiment 21. In a further embodiment of any one of embodiments 1 to20, further comprising at least one control device operably connected toone or more of: a) the first pump assembly; b) the sample injector; c)the first detector; d) the first fluidic routing unit; e) the secondpump assembly; and f) the second detector.

Embodiment 22. In one embodiment, the invention provides a method forseparating a sample comprising the steps of:

-   -   (i) capturing at least a portion of a sample on a trapping        column, said portion is obtained by separating the sample by        reversed-phase liquid chromatography (RPLC), said trapping        column comprising a stationary phase; and    -   (ii) subjecting the portion of the sample captured on the        trapping column to further separation by supercritical fluid        chromatography (SFC).

Embodiment 23. In a further embodiment of embodiment 22, furthercomprising separating the sample with a reversed-phase liquidchromatography comprising:

-   -   (i) introducing the sample into a first mobile phase;    -   (ii) driving the first mobile phase containing the sample        through a RPLC column; and    -   (iii) separating the sample on the RPLC column.

Embodiment 24. In a further embodiment of embodiment 23, furthercomprising detecting the presence of a component of the sample in thefirst mobile phase after passing through the RPLC column.

Embodiment 25. In a further embodiment of any one of embodiments 22 to24, further comprising eluting the portion of the sample captured on thetrapping column off the trapping column.

Embodiment 26. In a further embodiment of any one of embodiments 22 to25, further comprising detecting a component of the sample after furtherseparation by SFC.

Embodiment 27. In a further embodiment of any one of embodiments 22 to26, further comprising positioning a trapping column in a flow path ofthe first mobile phase downstream of the RPLC column for capturing atleast a portion of the sample separated by the RPLC column.

Embodiment 28. In a further embodiment of embodiment 27, furthercomprising switching the trapping column carrying the captured portionto a flow path of a second mobile phase for eluting the captured portionoff the trapping column.

Embodiment 29. In a further embodiment of embodiment 28, the step ofpositioning the trapping column in the flow path of the first mobilephase or the step of switching the trapping column to the flow path ofthe second mobile phase is performed in a fluidic routing unitinterfacing a fluidic path of the PRLC and a fluidic path of the SFC.

Embodiment 30. In a further embodiment of any one of embodiments 22 to29, further comprising re-capturing at least a portion of the sampleeluted off the trapping column on a focus column prior to furtherseparation by SFC.

Embodiment 31. In a further embodiment of any one of embodiments 22 to30, the first mobile phase flows through the trapping column in a firstdirection, and the portion of the sample captured on the trapping columnis eluted off the trapping column by flowing the second mobile phasethrough the trapping column in a direction opposite to the firstdirection.

Embodiment 32. In a further embodiment of any one of embodiments 22 to30, the first mobile phase flows through the trapping column in a firstdirection, and the portion of the sample captured on the trapping columnis eluted off the trapping column by flowing the second mobile phasethrough the trapping column in a direction same as the first direction.

Embodiment 33. In a further embodiment of embodiment 22, comprising thesteps of:

-   -   (i) introducing a sample into a first mobile phase;    -   (ii) driving the first mobile phase containing the sample        through a RPLC column;    -   (iii) separating the sample on the RPLC column;    -   (iv) detecting the presence of a component of the sample in the        first mobile phase after passing through the RPLC column;    -   (v) capturing on a first trapping column at least a first        portion of the sample separated on the RPLC column, said first        trapping column comprising a stationary phase;    -   (vi) eluting the first portion of the sample captured on the        first trapping column off the first trapping column;    -   (vii) subjecting the first portion of the sample captured on the        first trapping column to further separation by SFC; and    -   (viii) detecting a component of the sample after further        separation by SFC.

Embodiment 34. In a further embodiment of embodiment 33, furthercomprising the steps of:

-   -   (ix) capturing on a second trapping column at least a second        portion of the sample separated on the RPLC column, said second        trapping column comprising a stationary phase;    -   (x) eluting the second portion of the sample captured on the        second trapping column off the second trapping column;    -   (xi) subjecting the second portion of the sample captured on the        second trapping column to further separation by SFC.

Embodiment 35. In a further embodiment of any one of embodiments 22 to34, the RPLC column comprises a reversed-phase stationary phase.

Embodiment 36. In a further embodiment of any one of embodiments 22 to35, the stationary phase in the trapping column comprises areversed-phase material.

Embodiment 37. In a further embodiment of any one of embodiments 22 to36, further separation by SFC is performed on a SFC column comprising anormal phase stationary phase.

Embodiment 38. In a further embodiment of any one of embodiments 22 to36, further separation by SFC is performed on a SFC system comprising anarray of SFC columns.

Embodiment 39. In a further embodiment of embodiment 38, each of the SFCcolumns independently comprises a normal phase stationary phase.

Embodiment 40. In a further embodiment of embodiment 39, furthercomprising routing the portion of the sample captured on the trappingcolumn to a SFC column for further separation, said SFC column comprisesa stationary phase adapted for separating the components in the sample.

Embodiment 40A. In a further embodiment of embodiment 22, comprisingrouting the portion of the sample captured on the trapping column to aSFC column for further separation, said SFC column comprises astationary phase adapted for separating the components in the sample.

Embodiment 41. In one embodiment, the invention provides a method foranalyzing a sample using a chromatography system of Embodiment 1comprising: separating the complex sample into a first set of fractionsby reversed-phase liquid chromatography (RPLC) on the first separationunit; and further separating one or more of the fractions bysupercritical fluid chromatography (SFC) on the second separation unit.

Embodiment 42. In a further embodiment of embodiment 41, separation byRPLC on the first separation unit is based in part on a firstcharacteristic of the complex sample and separation by SFC on the secondseparation unit is based in part on a second characteristic of thecomplex sample, said second characteristic of the complex sample isdifferent from the first characteristic of the complex sample.

Embodiment 43. In a further embodiment of embodiment 41, the complexsample comprises a mixture of stereoisomeric components.

Embodiment 44. In a further embodiment of embodiment 43, thediastereomeric components are separated into one or more fractions byRPLC on the first separation unit, each said fraction comprising anenantiomeric pair.

Embodiment 45. In a further embodiment of embodiment 44, separation byRPLC on the first separation unit is based in part on the hydrophobicityof the complex sample.

Embodiment 46. In a further embodiment of embodiment 44 or 45, theenantiomeric pair is further separated into individual enantiomers bySFC on the second separation unit.

Embodiment 47. In a further embodiment of embodiment 46, separation bySFC on the second separation unit is based in part on the chirality ofthe complex sample.

Embodiment 48. In one embodiment, the invention provides a method forachiral-chiral analysis of a sample comprising a mixture ofstereoisomeric components using a chromatography system of Embodiment 1comprising: separating one or more diastereomeric component(s) ofinterest in the sample by RPLC on the first separation unit; andseparating the enantiomeric pair(s) of interest by SFC on the secondseparation unit in the same analytical run.

Embodiment 49. In a further embodiment of embodiment 48, furthercomprising determining an achiral purity based on a chromatogram fromthe RPLC separation and determining a chiral purity based on achromatogram from the SFC separation.

All references throughout, such as publications, patents, patentapplications and published patent applications, are incorporated hereinby reference in their entireties.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

What is claimed is:
 1. A method for separating a sample, the methodcomprising the steps of: (i) capturing a first mobile phase comprisingat least a portion of the sample on a trapping column, wherein the firstmobile phase is a reversed-phase liquid chromatography (RPLC) mobilephase, wherein said portion is obtained by separating the sample by aRPLC, and wherein said trapping column comprises a stationary phase; and(ii) subjecting the RPLC mobile phase comprising the portion of thesample captured on the trapping column to further separation by asupercritical fluid chromatography (SFC).
 2. The method of claim 1,further comprising separating the sample with the RPLC, wherein the RPLCcomprises: (i) introducing the sample into a first mobile phase; (ii)driving the first mobile phase containing the sample through a RPLCcolumn; and (iii) separating the sample on the RPLC column.
 3. Themethod of claim 2, further comprising detecting the presence of acomponent of the sample in the first mobile phase after passing throughthe RPLC column.
 4. The method of claim 2, wherein the RPLC columncomprises a reversed-phase stationary phase.
 5. The method of claim 1,further comprising eluting the portion of the sample captured on thetrapping column off the trapping column.
 6. The method of claim 1,further comprising detecting a component of the sample after furtherseparation by the SFC.
 7. The method of claim 1, further comprisingpositioning the trapping column in a flow path of the first mobile phasedownstream of a RPLC column for capturing the portion of the sampleseparated by the RPLC column.
 8. The method of claim 7, furthercomprising switching the trapping column carrying the captured portionto a flow path of a second mobile phase for eluting the captured portionoff the trapping column.
 9. The method of claim 8, wherein the step ofpositioning the trapping column in the flow path of the first mobilephase or the step of switching the trapping column to the flow path ofthe second mobile phase is performed in a fluidic routing unitinterfacing a fluidic path of the RPLC and a fluidic path of the SFC.10. The method of claim 1, further comprising re-capturing at least someof the portion of the sample eluted off the trapping column on a focuscolumn prior to further separation by the SFC.
 11. The method of claim1, wherein the first mobile phase flows through the trapping column in afirst direction, and the portion of the sample captured on the trappingcolumn is eluted off the trapping column by flowing a second mobilephase through the trapping column in a direction opposite to the firstdirection.
 12. The method of claim 1, wherein the first mobile phaseflows through the trapping column in a first direction, and the portionof the sample captured on the trapping column is eluted off the trappingcolumn by flowing a second mobile phase through the trapping column in adirection same as the first direction.
 13. The method of claim 1,comprising the steps of: (i) introducing a sample into the first mobilephase; (ii) driving the first mobile phase containing the sample througha RPLC column; (iii) separating the sample on the RPLC column; (iv)detecting the presence of a component of the sample in the first mobilephase after passing through the RPLC column; (v) capturing on thetrapping column at least a first portion of the sample separated on theRPLC column; (vi) eluting the first portion of the sample captured onthe trapping column off the trapping column; (vii) subjecting the firstportion of the sample captured on the trapping column to furtherseparation by the SFC; and (viii) detecting a component of the sampleafter further separation by the SFC.
 14. The method of claim 13, furthercomprising the steps of: (ix) capturing on a second trapping column atleast a second portion of the sample separated on the RPLC column, saidsecond trapping column comprising a stationary phase; (x) eluting thesecond portion of the sample captured on the second trapping column offthe second trapping column; (xi) subjecting the second portion of thesample captured on the second trapping column to further separation bythe SFC.
 15. The method of claim 1, wherein the stationary phase in thetrapping column comprises a reversed-phase material.
 16. The method ofclaim 1, wherein further separation by SFC is performed on a SFC columncomprising a normal phase stationary phase.
 17. The method of claim 1,wherein further separation by the SFC is performed on a SFC systemcomprising an array of SFC columns.
 18. The method of claim 17, whereineach of the SFC columns independently comprises a normal phasestationary phase.
 19. The method of claim 18, further comprising routingthe portion of the sample captured on the trapping column to a SFCcolumn for further separation, said SFC column comprises a stationaryphase adapted for separating components in the sample.
 20. The method ofclaim 1, wherein the volume of the RPLC mobile phase comprising theportion of the sample subjected to further separation by the SFC isabout 10 μL to about 100 μL.
 21. The method of claim 1, wherein thevolume of the RPLC mobile phase comprising the portion of the samplesubjected to further separation by the SFC is about 30 μL or less.
 22. Amethod for analyzing a sample using a chromatography system, the methodcomprising: separating the sample into a first set of fractions by areversed-phase liquid chromatography (RPLC) on a first separation unit;and further separating one or more of the fractions by a supercriticalfluid chromatography (SFC) on a second separation unit, wherein thechromatography system comprises a first fluidic routing unit comprisinga plurality of sample loops, said first fluidic routing unit isconnected to the first separation unit and the second separation unit,wherein at least one of the plurality of sample loops comprises atrapping column, said trapping column comprising a stationary phase, andwherein the chromatography system is configured to analyze the sampleusing the chromatography system by for first separating the sample inthe first separation unit and subsequently introducing at least aportion of the sample eluted from the RPLC column of the firstseparation unit to the second separation unit.
 23. The method of claim22, wherein separation by the RPLC on the first separation unit is basedin part on a first characteristic of the sample and separation by theSFC on the second separation unit is based in part on a secondcharacteristic of the sample, said second characteristic of the sampleis different from the first characteristic of the sample.
 24. The methodof claim 22, wherein the sample comprises a mixture of stereoisomericcomponents.
 25. The method of claim 24, wherein the stereoisomericcomponents are separated into one or more fractions by the RPLC on thefirst separation unit, each of said fraction comprising an enantiomericpair.
 26. The method of claim 25, wherein separation by the RPLC on thefirst separation unit is based in part on the hydrophobicity of thecomplex sample.
 27. The method of claim 25, wherein the enantiomericpair is further separated into individual enantiomers by the SFC on thesecond separation unit.
 28. The method of claim 27, wherein separationby the SFC on the second separation unit is based in part on thechirality of the sample.
 29. A method for achiral-chiral analysis of asample comprising a mixture of stereoisomeric components using achromatography system, the method comprising: separating one or morediastereomeric component(s) of interest in the sample by areversed-phased liquid chromatography (RPLC) on a first separation unit;and separating the enantiomeric pair(s) of interest by a supercriticalfluid chromatography (SFC) on a second separation unit in the sameanalytical run, wherein the chromatography system comprises a firstfluidic routing unit comprising a plurality of sample loops, said firstfluidic routing unit is connected to the first separation unit and thesecond separation unit, wherein at least one of the plurality of sampleloops comprises a trapping column, said trapping column comprising astationary phase, and wherein the chromatography system is configured toperform achiral-chiral analysis of the sample by for first separatingthe sample in the first separation unit and subsequently introducing atleast a portion of the sample eluted from the RPLC column of the firstseparation unit to the second separation unit.
 30. The method of claim29, further comprising determining an achiral purity based on achromatogram from the RPLC separation and determining a chiral puritybased on a chromatogram from the SFC separation.